AU4888700A - Protein particles for therapeutic and diagnostic use - Google Patents

Description

S&F Ref: 396808D1

AUSTRALIA

PATENTS ACT 1990 COMPLETE SPECIFICATION FOR A STANDARD PATENT

ORIGINAL

Name and Address of Applicant Actual Inventor(s): Address for Service: Invention Title: HemoSphere, Inc.

15251 Alton Parkway Irvine California 92718 United States of America Richard C. K. Yen Spruson Ferguson St Martins Tower 31 Market Street Sydney NSW 2000 Protein Particles for Therapeutic and Diagnostic Use The following statement is a full description of this invention, including the best method of performing it known to me/us:- IP Australia Documents received on: 2 8 JouL 2000 Batch No: 5845c Protein Particles for Therapeutic and Diagnostic Use Background of the Invention Therapeutic drugs are typically administered orally or by intramuscular, subcutaneous, intraperitoneal, or intravenous injections. Intravenous injection is the most direct means of administration and results in the fastest equilibration of the drug peak serum levels within a short time, however. Toxic effects can result from such high levels, especially if the drug is given as a bolus injection. To avoid such high concentrations, drugs can be administered slowly as a continuous drip. This however requires prolonged nursing care and, in some cases, hospitalization which itself entails high cost. To avoid this, efforts have been made to 0o develop means of administering drugs within stable carriers which allow bolus intravenous injections but provide a gradual release of the drugs inside the vasculature.

The reticuloendothelial system (RES) directs drugs preferentially to the liver and spleen, and its uptake of a carrier thus interferes with the distribution of the drug to other parts of the body. If however the carriers are small enough so that the phagocytic cells such i15 as macrophages do not preferentially ingest them, the carriers would escape the RES long enough to perform other tasks. If the carriers also contain antibodies or other ligands on their surfaces which e [R:\LBUU]27628.doc:mcc specifically bind to antigenic sites or specific receptors, these antibodies or ligands will direct the drugs to specific cell types containing these sites or receptors. This would result in a higher concentration of the drug near the surfaces of the targeted cells without a higher risk of systemic side effects.

Entrapment of useful agents serves useful purposes in other medical applications as well. Tiny air bubbles, for example, are useful in ultrasonography, where they are used to provide strong contrast to blood vessels and organs traversed by the bubbles. If the bubbles are injected through a peripheral vein, however, they must travel through the right heart, the pulmonary vasculature and then the left heart before they can reach to the other internal organs. Since the bubbles are inherently unstable, they are not able to remain small enough for effective ultrasonographic contrast by the time the intended organs are reached. Entrapment of small air bubbles "in small particulate carriers would allow the bubbles to serve ."their intended function even after long distances of travel S 20 within the intravascular compartment.

Similar advantages by using small particulate carriers for contrast material for*CAT scans and nuclear magnetic resonance scans. Abnormally high concentrations of contrast material at an injection site which might lead to false interpretation of the results could be avoided by administration of the contrast material as an agent retained in a particulate carrier to be released later at the site of the organ of interest.

Oxygen is another vital biological molecule that can be 0 carried within a particulate carrier if the carrier contains hemoglobin. While hemoglobin molecules in large amounts are toxic to the human body, entrapment of hemoglobin within a particulate carrier will reduce its toxicity to vital organs while permitting it to deliver oxygen.

To summarize, stable porous and membraneless carriers which deliver biological agents to sites within the body offer many advantages. The two major approaches of particulate carriers in the prior art are liposomes and microspheres.

In liposomes, a shell is formed by a lipid layer or multiple lipid layers surrounding a central hydrophilic solution containing the medication. The lipid layers are inherently unstable and much research went into stabilizing them during the manufacturing process. In addition, the lipid layer(s) may serve as a barrier to diffusion of certain molecules. It is difficult for a hydrophilic substrate to diffuse through the hydrophobic layers into the interior of the liposomes, or conversely, for the drugs to be released without physical destruction of the lipid layer(s).

Microspheres, in contrast to liposomes, do not have a surface membrane or a special outer layer to maintain their intactness. Most microspheres are more or less homogenous in structure. To maintain the stability of the microspheres, manufacturing procedures in the prior art include a cross-linking process to stabilize the microspheric mass. The cross-linking agent however alters the chemical nature of the natural biological molecule, which may render the resultant product antigenic to the injected host. An anaphylactic reaction to such a newly created antigenicity is unpredictable and potentially dangerous.

Protein particles in essentially spheric form are useful in the encapsulation and delivery of nutrients and biologics such as oxygen, enzymes, drugs, and information molecules (DNA, RNA and hybrid molecules of DNA and RNA) to cells and tissues.

20 To preserve the intactness of the spheres after synthesis and to allow further purification or concentration of the spheres, a variety of methods have been used during or after synthesis to prevent resolubilization of the protein particles.

These methods include heat denaturization (see, Evans, et al.

U.S. Patent No. 3,663,685 and Widder, et al., Adv. Pharmacol.

and Chemother. 16:213-271 (1979)); addition of a cross-linking agent to initiate and complete formation of particles composed of covalently and irreversibly crosslinked protein molecules (see Oppenheim, U.S. Patent No. 4,107,288); and the addition of a cross-linking agent following the formation of protein spheres in the presence of alcohol (see Yen, U.S. Patent No.

5,069,936). More recently, a method has been described for stabilizing protein spheres against resolubilization by incorporating hemoglobin molecules into albumin spheres (see Yen, co-pending application serial no. 08/212,546.

Other literature of potential relevance to the present invention is as follows.

United States Patent No. 4,269,821, Kreuter, et al., May 26, 1981, for "Biological Material" discloses processes for the preparation of submicroscopic particles of a physiologically acceptable polymer associated with a biologically active material by using a cross-linking agent such as a polymerisable material soluble in a liquid medium (methyl methacrylate as an example).

United States Patent No. 3,663,685, Evans et al., May 16, 1972, for "Biodegradable Radioactive Particles" (hereafter "Evans") discloses a method of preparing biodegradable radioactive particles by using heated water-oil solutions.

Widder, et al., "Magnetically Responsive Microspheres And Other Carriers For The Biophysical Targeting Of Antitumor Agents", Advances in Pharmacology and Chemotherapy 16:213-271 (1979) disclose emulsion polymerization methods of preparation of albumin microspheres (pages 233-235) and preparation of magnetically responsive albumin microsphere (pages 241-250).

The methods essentially involve emulsification and heat denaturation of a water-oil solution-to produce and stabilize microspheres. The authors also state that for heat sensitive drugs the microspheres are stabilized by chemical cross-linking.

To summarize this literature, typical prior art processes require irradiation, heat, or reaction with a cross-linking agent to polymerize the "monomers" (which are the individual protein molecules such as human serum albumin or gelatin 20 molecules) to convert them to stable particles. Prior art methods which use heat to cross-link and stabilize the protein involve irreversible denaturation of the proteins which renders them "foreign" to the host body.

United States Patent No. 5,049,322, Devissaguet, et al., 25 Sep. 17, 1991 discloses a method of producing a colloidal system containing 150-450 nm particles by dissolving a protein ingredient in a solvent and adding ethanol or mixture of ethanol containing surfactant. Devissaguet does not disclose adding a second protein ingredient. Devissaguet discloses a process of producing colloidal spheres which have a distinct "wall" (column 2, line 25) or "layer" (column 8, line 33) of *substance A which is different from the "core" of substance B (column 8, line 18), where the substance B may be a biologically active substance. This disclosure requires that the wall material and the core material both be present in a first liquid phase, which is then added to a second liquid phase that is a non-solvent for both materials. The resulting product is not homogeneous, and relies on the wall for its particle integrity.

Albert L. Lehninger, Biochemistry: The Molecular Basis of Cell Structure and Function (1972) discloses that ethanol as a solvent can decrease the ionization of proteins and therefore promote their coalescence and produce "colloidal suspensions".

Lehninger does not disclose a special method of preparing colloidal suspensions, but rather generally a method of promoting protein coalescence by using ethanol, "[s]ince a decrease in dielectric constant increases the attractive force between two opposite charges, ethanol decreases the ionization of proteins and thus promotes their coalescence" (page 134, lines 21 through 25, citations omitted). Lehninger has defined the process of "coalescence" as a process leading to "insoluble aggregates" (page 133, lines 31 through "Remington's Pharmaceutical Sciences", 7th ed. (1985) discloses some general knowledge of "colloidal dispersions".

Remington teaches that adding surfactant "stabilizes the dispersion against coagulation" (page 286, column 2, lines 59 and 60), where the surfactant molecules "arrange themselves at the interface between water and an organic solid or liquid of low polarity in such a way that the hydrocarbon chain is in contact with the surface of the solid particle or sticks inside the oil droplet while the polar bead group is oriented towards the water phase" (page 286, column 2, lines 30 through Remington does not specially disclose the use of any particular 20 protein molecules such as globin as the primary protein.

SUMMARY OF THE INVENTION It has now been discovered that protein particles in the nanometer and micrometer size range, suspended in an aqueous medium, can be made stable against resolubilization 25 prevented from redissolving) upon storage, dilution and dialysis, by the inclusion of certain noncrosslinking additives, and that noncrosslinking additives can also reduce or eliminate the tendency of protein particles to aggregate in aqueous suspensions. In particular, particles of non- 30 crosslinked and non-denatured albumin in a suspension are stabilized against resolubilization by the inclusion of reducing agents, oxidizing agents, phosphorylated compounds, sulfur containing compounds, polymers and combinations thereof.

A convenient method of forming the particles of either protein is by adding a water-soluble lower alkyl alcohol to an aqueous solution of the protein. Upon formation of the particles, the solution turns turbid. The stabilizing agent may then be added in a volume sufficiently small so that the aqueous solvent medium (for the agent) will not cause a decrease in the alcohol concentration that will disrupt the protein particles. After a minimal time of interaction which allows the stabilizing agent to completely stabilize the protein spheres against resolubilization, the suspension may be diluted in an alcohol-free aqueous medium to lower the alcohol content. Alternatively, the suspension may be dialyzed against an aqueous medium to remove any molecular species small enough to pass through the dialysis membrane alcohol, surfactant, and other added but unincorporated reagents) or washed by repeated cycles of centrifugation and resuspension of the pelleted protein particles in a new medium. Even if neither dilution or dialysis is performed and the particle suspension is administered as formed, a similar effect occurs when the suspension is administered to a patient where it combines with the patient's serum or other bodily fluids. In any case, both dilution and dialysis raise the tendency of the particles to return to solution. In a laboratory vessel, resolubilization is evident when the turbidity disappears and is replaced once again by a clear solution. The additive prevents this from happening without the need for crosslinking.

The benefits of the particulate form are thus retained without the need for a crosslinking reaction or the danger of an 20 irreversibly crosslinked particle.

Aggregation of the particles arises in some cases immediately upon their formation and, in some cases, upon dialysis or storage for several hours. Aggregated particles are often too large to be administered effectively, and when 25 close control of the particle size is desired, this is defeated by aggregation. The discovery that aggregation can be avoided by the inclusion of the additives listed above therefore adds a further benefit to the benefits achieved by the elimination of crosslinking.

This invention therefore permits the formation of albumin particles in the nanometer to micrometer size range, in a form S. closer to their natural form than the forms of the prior art.

The particles thus constitute a more closely controlled agent for in vivo administration, either for their own administration or as a vehicle for other therapeutic or diagnostic agents, or as a building block for cellular processes.

One example of therapeutic use is to inject or infuse fibrinogen coated particles intravenously for the purpose of decreasing bleeding time in thrombocytopenic patients or animals. Thrombocytopenic animals lack a sufficient concentration of platelets which are essential cellular elements responsible for hemostasis. The key event in control of bleeding is the activation of platelets at the site of a wound, which leads to binding of fibrinogen onto the surface of platelets. Normally, after the activation of platelets chemicals are released from activated platelets to activate other platelets in the vicinity of the wound to quickly aggregate to form a plug to stop bleeding. In addition, the fibrinogen on the surface of the platelets participates in the clotting factor cascade that causes the soluble factors in the blood to also form a plug. However, in a thrombocytopenic animal, the number of platelets is not sufficient to form a plug quickly. As a result it takes a much longer time for bleeding to stop. By infusing a suspension of fibrinogen coated synthetic spheres or particles, the total number of solid-bound fibrinogen molecules were increased in the blood to result in improvement in bleeding time and a decrease in the amount of blood loss. In one embodiment of the invention, cross linked albumin particles coated with fibrinogen were used. It is anticipated that in patients about to undergo surgery with major blood loss, or in trauma patients such as soldiers wounded in the battlefield, even though they have a "normal" platelet, count, an augmentation of the number of 20 fibrinogen coated particles will decrease blood loss and lead to shortened surgical time.

These and other features and advantages of the invention will be apparent from the description which follows.

DETAILED DESCRIPTION OF THE INVENTION 25 AND PREFERRED EMBODIMENTS The protein particles which are the subject of this invention are monodisperse particles, generally spherical in shape. The term "monodisperse" as used herein denotes discrete single particles which are individually suspended in the 30 aqueous suspension and are neither attached nor adhered to other particles, as distinct from aggregates or aggregated particles, which are groups of two or more, and as many as a hundred or more, such particles adhering to each other by surface interaction or attraction',.the aggregates themselves being suspended in the medium in the same manner as the monodisperse particles. While large aggregates can be discerned by the naked eye, a microscope is generally required to differentiate mid-size to small aggregates from monodisperse particles.

Agents which were found to have a stabilizing effect on protein particles include reducing agents, oxidizing agents, high molecular weight polymers polyethylene glycol), hydrogen acceptor molecules NADP), carboxylic acids having multiple oxygen functionality, and sulfur containing ring compounds thioctic acid).

More particularly, the stabilizing agents which are reducing agents can be either organic reducing agents such as dithiothreitol or mercaptoethanol, or inorganic reducing agents such as sodium sulfite or sodium bisulfite.

Stabilizing agents which are high molecular weight polymers will typically be polyethyleneglycols.

An example of a stabilizing agent which is a hydrogen acceptor molecule is NADP.

Yet another group of stabilizing agents are those which can be classified as sulfur containing ring compounds, an example of which is thioctic acid.

Still yet another group of stabilizing agents are those which have multiple oxygen functionality, particularly alphaketo carboxylic acids, alpha-hydroxy carboxylic acids and dicarboxylic acids. Examples in this group include lactic acid (D and L forms), succinic acid, ascorbic acid and 1- 20 ketoglutaric acid.

The stabilizing agents can either be present in the protein solution before the addition of alcohol, such that particles subsequently formed are stable against resolubilization, or be added to suspensions of protein 25 particles. Similarly, biological molecules can be added to the protein solution before formation of the particles or spheres, or they can be added after the appearance of turbidity which indicates the formation of particulates. The biological molecules are carried either in the interior or on the surface of the spheres, or both. Additionally, the properties of such added molecules are not altered by the stabilizing agents.

The size range of the particles of the present invention extends into both nanometer and micrometer ranges. In general, particles of interest will primarily range from about 50 to about 5000 nanometers in diameter, in monodisperse form. The appropriate or optimal size range for particular uses of the particles or methods of administration will vary with the use or method.

The aqueous medium in which the particles are formed is a homogeneous, water-containing liquid, which may also contain additional components such as surface active agents, buffering agents and inorganic ions. Aqueous media of particular interest in the context of this invention are distilled or deionized water, normal saline and hypotonic saline. In preferred embodiments of the invention, the aqueous medium in which the particles are formed further includes the alcohol which induces the turbidity, the alcohol being fully miscible with the water in the medium to result in a homogeneous continuous phase. In most applications of the invention, the particles will constitute at least about 1.0 g per liter of the suspension, preferably from about 1.0 g per liter to about 150 g per liter, and in many applications at least about 3.0 g per liter, and preferably from about 5.0 g per liter to about 50 g per liter.

In embodiments of the invention in which the suspension is subjected to dilution in, or dialysis against, a second aqueous medium, the second aqueous medium will also be a watercontaining liquid, most likely containing neither alcohol nor surfactants. The second aqueous medium is alcohol-free, and is preferably a biological fluid, a fluid similar in composition o to a biological fluid, or a fluid which is compatible with a 9 biological fluid. Compatible fluids are those which do not cause adverse physiological effects upon administration.

20 Examples are water, normal saline, and 5% aqueous human serum albumin (HSA).

Dilution may be done to varying degrees, although in most cases the amount of aqueous medium added will result in a S volume increase of at least about 50%. The invention is 25 particularly effective when dilutions are performed by adding an equal volume of aqueous medium (100% volume increase) or greater.

The alcohol referred to above is a lower alkyl alcohol, preferably either methanol, ethanol, n-propanol, isopropanol or 0 n-butanol. Among these alcohols, ethanol and n-butanol are particularly preferred. When included, the alcohol is present in an amount sufficient to induce turbidity in the initial aqueous solution of the protein, and preferably to cause precipitation of all protein dissolved in the solution. In most applications, this amount will fall within the range of about 5% to about 80% by volume of the aqueous medium, and preferably from about 10% to about The primary protein component of the particles of interest in the present invention is albumin, which is neither denatured nor crosslinked. The albumin, the albumin may be any of the various known types of albumin, the choice being governed by the route or method of administration to the patient. Serum albumin, particularly human serum albumin, is preferred.

The surfactants used in certain embodiments of the invention are anionic water-soluble surfactants, preferably sodium or potassium alcohol sulfates. Particularly preferred are sodium or potassium C 6

-C

16 alkyl sulfates and sodium or potassium C 8

-C

14 alkyl sulfates. Sodium lauryl sulfate and sodium tetradecylsulfate are the most preferred.

The amount of surfactant'used in these embodiments may vary depending on the other system parameters. For albuminbased particles where the particles are formed as a suspension in a first aqueous medium, then diluted by addition of a second aqueous medium, best results will be obtained using at least about 1.0 g of surfactant per liter of the suspension prior to dilution or dialysis. Preferably, the surfactant constitutes from about 0.5 g to about 5 g per liter of the suspension, particularly when the suspension contains at least about 15 g of particles per liter of suspension.

The following examples are offered solely for purposes of illustration, and are intended neither to limit nor to define the invention.

EXAMPLE 1 S:i" This example illustrates the synthesis of particles of human serum albumin (HSA), followed by dilution in either water or normal saline, the particles being formed without the inclusion of a stabilizing agent against resolubilization upon dilution, but with the use of sodium lauryl sulfate (SLS) to prevent aggregation of the particles; The HSA was prepared by diluting stock HSA (25% in normal saline) in distilled water to 80 mg/mL. Mixtures of this solution were then prepared by combining it with water and SLS 30 in the amounts shown in Table I, followed by the addition of ethanol. As shown in the table, turbidity resulted in each tube, with the particles in tubes 30 and 31 (containing 2 mg/mL and 1 mg/mL, respectively, of the SLS based on the solution prior to the addition of the ethanol) being monodisperse and those in tube 32 (lacking SLS) being aggregated. The table also shows that upon dilution of the tubes with equal volumes of distilled water or normal saline buffer, the contents of tubes 30 and 31 redissolved into a clear solution within one hour.

These results indicate that a stabilizing agent is needed for HSA particles to prevent redissolving of the particles upon dilution, and a surfactant is needed to prevent the particles from aggregating.

TABLE I HSA Particles with Surfactant but no Stabilizing Agent Tube Contents (balance: water to achieve total volume of mL before addition of alcohol) Size (and Condition) of HSA SLS Particles Tube (mL at 80 (mL at 8 Ethanol Formed Effect of No. mg/mL) mg/mL) (mL) (im) Dilution 0.25 0.250 0.8 0.1 (mono- redissolves disperse) 31 0.25 0.125 0.8 0.05 (mono- redissolves disperse) 32 0.25 0.000 0.8 (aggregated) e ••oo o og o• ft 10 EXAMPLE 2 This example illustrates the use of a stabilizing agent to prevent resolubilization of human serum albumin (HSA) spheres.

Ethanol (70% in water) was added dropwise to a solution of HSA (150 mg/mL in normal saline, in a volume of from about 1.2 to 1.8 times the volume of protein solution, to form a turbid suspension of spheres. The average diameter of the spheres examined under light microscopy was about 0.8 to 1.2 microns. When the HSA concentration is 50 mg/mL or lower, the spheres or particles formed are typically 0.2 to 0.5 microns in 20 diameter.

Aliquots of the suspension were dispensed into tubes mL per tube). A stock solution of stabilizing agent such as a reducing agent (1M of sodium bisulfite) was serially diluted two fold each step for 12 steps. Then 5 tL of the stabilizing agent at various concentrations was added to 0.5 mL of unstabilized sphere suspensions. Five microliters of N.S. was added to each of the control tubes. The protein particles remain intact at this level of dilution (typically 1% dilution). Larger volumes of any non-alcohol containing aqueous media greater than 2 volumes per 100 volumes of nonstabilized protein particle suspensions) will increase the risk of partial resolubilization of the protein particles, resulting in a lower concentration of particles which had approximately the same size range.

After 2 hours, an aliquot from each tube was examined under the microscope to check for any change in the average diameter of the spheres or the presence of aggregates. None were observed.

After incubation for at least 2 hours, normal saline mL) was added to each tube. Control tubes and HSA particle suspensions with insufficient amount of stabilizing agent turned from turbid suspensions to clear solutions within minutes. The minimum final concentration of stabilizing agents (before addition of the non-alcohol dilution medium) needed to maintain turbidity after dilution of the alcohol content was noted. The highest final concentration of stabilizing agents (before addition of the non-alcohol dilution medium) which allow the particles to remain monodisperse without the formation of aggregates was also noted.

After the approximate effective concentrations (the range between the maximal and minimal final concentrations) from the twofold dilutions of stock stabilizing agents were found, the *"experiments were repeated with narrower ranges of final ."concentrations to further refine the effective range accurately.

Additional agents were checked for their ability to stabilize HSA spheres against resolubilization. The effect of a longer incubation times (up to 18 hr) was also examined. It was found that longer incubation times allowed a lower concentration of stabilizing agent to be used while retaining effectiveness in preventing resolubilization of the spheres, 0 without aggregate formation.

Table II provides the minimal and maximal final .:.concentrations of each stabilizing agent in the final volume of sphere suspension (before addition of the non-alcohol dilution medium) that was needed to maintain particulate intactness against resolubilization without causing aggregate formation.

TABLE II Effective Concentrations of Agents Used to Stabilize Protein Spheres Effective Concentrations

(AM)

Agent Minimum Maximum r Sodium Bisulfite* (NaHSO 3 Sodium Sulfide*

NADP*

DTT*

Glutathione* (reduced form) PEG (MW 2000)* PEG (MW 5000)* PEG (MW 8000)* Sodium Bisulfite Sodium Sulfite (Na 2

SO

3 2-Mercaptoethanol DL-Lactic Acid Thioctic Acid (oxidized) Stannous Chloride Succinic Acid Ascorbic Acid 1-Ketoglutaric Acid Cysteine Manganese Chloride 650 500 250 100 100 12.5 12.5 12.5 50 50 10** 10 500 10 250 1000 1000 500 500 1000 5000 2500 2500 1000 2500** 2500Mg/mL 2500 pg/mL 2500 g/mL 500 1000 5000 1000 500 2500 2500 2500 1000 2500 2500 .e *Incubation time between agent and sphere suspension was 2 hours, all other incubation times were approximately 18 hours.

Lowest final concentration tested was 10 MM. ***Highest final concentration tested was 2500 MM.

4* Initial conc of HSA was instead of 15% which required approximately double the minimum concentration of agent to achieve stability against resolubilization.

EXAMPLE 3 This example illustrates particular agents that were not effective in protecting spheres against resolubilization.

The same experimental protocol was followed as described in Example 2. Turbidity of the suspensions was reversed to clear solutions within 2 hours of dilution with normal saline (3 volumes per volume of suspension.) Table III provides the unsuccessful agents used.

TABLE III Ineffective Stabilizing Agents Final Concentration (pM, unless Agent specified) r r r lowest highes t 10 10,000 NaNO 2 (sodium nitrite)

NADH

NADPH

Glutathione (oxidized form) Hydrogen peroxide Sodium Glucuronate Isocitric 2-Hydroxybutyric Acid Glucose Fructose Lactose Galactose Cytochrome C Phenazine Methosulfate Dichlorophenolindophe nol 50 10,000 50 10,000 50 10,000 0.01% 10 10 10 10 10 0.3% 10,000 10,000 10,000 10,000 10,000 10,000 10,000 10 10,000 100 500 FAD 10 FMN 10 1000 Triphenyltetrazolium 10 1000 Chloride EXAMPLE 4 This example illustrates the minimal time of interaction between sphere suspension and stabilizing agent needed for stabilization of spheres.

Sodium bisulfite was selected as a typical stabilizing agent for use in this experiment. Protein spheres were formed as described in Example 2. Two concentrations of stock sodium bisulfite (0.05 and 0.1 M) were used (5 ML added per mL of sphere suspension). Thereafter at the indicated times, 1.5 mL of N.S. was added to evaluate the effect of various incubation times on sphere stability. The results are provided in Table

IV.

TABLE IV Interaction Times to Stabilize Protein Spheres AGENT INTERACTION FINAL CONCENTRATION OF AGENT TIME (hr) 500IM 10001M Sodium Bisulfite Time for solution to clarify 0.17 <1 min 1 min 0.33 1 min 3 min 0.50 5 min 1 hr 1.00 2 hr Turbid overnight 2 to 6 Turbid overnight Turbid overnight As can be seen from the results in Table IV, stability was not obtained with interaction time less than approximately minutes. Additionally, a higher concentration of stabilizing agent is more effective in stabilizing spheres within a shorter time of interaction. In general, incubation of at least two hours will be needed. This shows that the stabilization process is a gradual one, suggesting a physical rearrangement of molecules within or on the surface of the spheres to render the spheres gradually more resistant to resolubilization.

EXAMPLE This example illustrates that premixing a stabilizing agent with HSA solution before the addition of alcohol results in stable spheres.

To each of a series of tubes containing 1.0 mL of HSA solution (15% in normal saline), was added 30 tL of one of the following agents: sodium bisulfite (0.025 to 0.25 DTT (0.025 to 0.1 NADP (0.05 to 0.1 thioctic acid (oxidized, 0.01 to 0.025 M) and control solution (normal saline).

Ethanol was then added dropwise (approximately 1.7 to 1.88 mL) to form turbid suspensions. After 2 hours at room temperature, 5 mL of normal saline was added to see if the turbid suspensions became clear solutions. Only the control tube which had no stabilizing agent added before formation of the spheres reverted to a clear solution within 5 minutes. All other tubes were turbid after more than 2 hours.

Ethanol (70% in water) was added dropwise to a parallel set of tubes containing 1.0 mL of HSA solution (15% in normal saline) to form a turbid suspension, followed by the addition 20 of 30 AL of the above mentioned agents at the above mentioned concentrations. After 2 hours at room temperature, 5 mL of normal saline was added to each tube. Again, only the control tube which had no stabilizing agent added after formation of the spheres reverted to a clear solution within 5 minutes. All other tubes were turbid after more than 2 hours. This result indicates that there is no significant difference between adding the stabilizing agent before or after the formation of the spheres.

To a third parallel set of tubes, 30 AL of the above 30 mentioned agents were added to 1.0 mL of HSA without addition of alcohol. No turbidity was seen during or after the two hours observed. This result indicates that the agents only stabilized the spheres and did not by themselves cause the formation of spheres.

EXAMPLE 6 This example illustrates that protein particles can be stabilized by a hydrogen-accepting agent in the presence of a hydrogen-donating agent.

We have shown in the above experiments that a hydrogenaccepting (oxidized state) agent such as NADP can stabilize protein particles against resolubilization but'that a similar molecule in the reduced state (NADPH) cannot.

Ethanol (70% in water.) was added to HSA solution to produce turbidity. To aliquots of 1.0 mL of protein particle suspension was added 5 pL of the agent(s) (in molar concentrations) listed in Table V. After 2 hours, 5 mL of N.S.

was added to each tube and the suspension was examined for turbidity.

4.

4 .4 .4 4.

TABLE V Stabilization of Protein Particles Using Combinations of Hydrogen-Accepting and Hydrogen-Donating Agents Tube NADP NADPH After addition of

N.S.

21 0 0 clear 22 0 0.1 clear 23 0.1 0 turbid 24 0.1 0.10 turbid 0.1 0.25 turbid 26 0.1 0.50 turbid As Table V indicates, a hydrogen-accepting agent (NADP) is effective in.stabilizing protein spheres in the presence or absence of a hydrogen-donating agent (NADPH).

EXAMPLE 7 15 This example illustrates that the addition of chemotherapeutic agents does not interfere with sphere formation or stabilization.

Adriamycin was added to HSA to result in a concentration of 0.1 mg/mL. Control HSA was 15% without 20 adriamycin.

Ethanol was added to the HSA solutions until the solutions became turbid. Thereafter, 10 pL of sodium bisulfite (0.05 M to 0.5 M) was added per 1.0 mL of suspension. After 2 hours incubation, 5 mL of N.S. was added and the mixtures were examined for resolubilization of the protein spheres.

The results showed that both control spheres and spheres with adriamycin were stable against resolubilization.

Incorporation of adriamycin in the spheres did not interfere with the action of the stabilizing agent.

EXAMPLE 8 This example illustrates that enzymes incorporated within the interior or on the surface of the protein particles, are still active after the particles have been stabilized by the addition of a stabilizing agent.

To demonstrate that enzymes trapped within the interior and on the surface of protein particles stabilized with a stabilizing agent can retain their catalytic function, 5 gL of a commercial preparation of peroxidase (Horse Radish Peroxidase Type VI, Sigma Chemical Co., St. Louis, MO) was added to 1 mL of a HSA solution Ethanol was subsequently added to,produce protein spheres. Within 5 minutes, 5 iL of sodium bisulfite (0.1 M) was added to 1.0 mL of the suspension to stabilize the spheres. After overnight incubation, the spheres were washed three times in normal saline (10 mL each time) without resolubilization.

A solution containing a peroxidase substrate was added to the sphere suspension. A strongly positive reaction (read at 490 nm wavelength) was produced within 5 min. As a control, a portion of the supernatant from the final wash was incubated with the peroxidase substrate. The reaction was negative, indicating that the peroxidase reactivity observed resided with the spheres and not from any residual enzyme in the supernatant.

This experiment did not prove whether all the enzymes were inside the spheres or whether some were exposed on the surface of the particles. However, the addition of a stabilizing agent S, did not result in substantial loss of enzyme activity.

20 EXAMPLE 9 .This example illustrates that antigenic sites of proteins within the interior and surface of spheres remain unchanged for reaction with the specific antibody even after stabilization of the spheres with a stabilizing agent.

o 25 Rabbit IgG was chosen as the antigen. A goat anti-rabbit ***(GAR)IgG (conjugated to peroxidase) was used as the antibody.

The protocol in Example 9 was followed except that rabbit IgG was incorporated within the spheres and on their surfaces instead of the peroxidase enzyme.

The rabbit IgG-containing spheres were washed three times in normal saline (10 mL each time), and a diluted aliquot of GAR was added to the sphere suspension. After incubation at 37C for 1 hr, unreacted GAR was removed by three additional washes in normal saline (10 mL each). The supernatant of the last wash was checked for any residual GAR activity, which may represent excess GAR, or leakage of GAR complexed with rabbit IgG that resulted from partial resolubilization of the particulates.

The result showed that the spheres, but not the supernatant, had peroxidase activity. This experiment suggested that the spheres may be porous enough for some GAR to penetrate the surface of the spheres to bind to the rabbit IgG inside the spheres. Alternatively, enough rabbit IgG antigenic sites may be exposed on the surface of the spheres to allow enough GAR binding for easy detection.

EXAMPLE This example illustrates that antibodies are still reactive within the interior and surface of the albumin spheres even after stabilization of the spheres by a stabilizing agent.

The protocol of Example 9 was used except that an antibody was incorporated instead of an antigen. As an example of antibody, a commercial source of polyclonal goat anti-human fibrinogen IgG was used. The suspension of antibody-containing spheres (1.0 mL) was incubated overnight with sodium bisulfite AL of a 0.05 M solution). The resulting particulates were washed three times with normal saline (10 mL each) without resolubilization. Control spheres were similarly prepared except without any incorporated antibody.

The sphere pellets were then resuspended in a human fibrinogen solution (0.01 mg/mL) for 2 hr at room temperature, after which the spheres were washed again twice to remove excess fibrinogen (the antigen).

For detection of antigen binding, a mouse anti-human fibrinogen (MAH) monoclonal antibody was used to react with the spheres for 2 hours, followed by removal of the MAH by washing twice. Subsequently, a sheep anti-mouse IgG (conjugated to peroxidase) was used to detect the presence of mouse monoclonal antibody-human fibrinogen complex on or within the spheres.

The result showed that the supernatant of the last wash was negative, but the spheres showed positive reactivity indicating that the primary antibody (goat anti-human fibrinogen IgG) was present within or on the surface of the spheres even after multiple washings.

EXAMPLE 11 This example illustrates that enzymes, antigens and antibodies can be added during the stabilization phase to spheres without loss of activity.

The protocol of Examples 8, 9 and 10 for enzyme, antigen and antibody were repeated, respectively, except that spheres were first formed without the respective biological agent. The respective biological agents were added within 30 seconds of the addition of the stabilizing agent. All the subsequent washings and reagents used were identical.

The results again showed that enzymes, antigens and antibodies could be stabilized essentially on the surfaces of the spheres during the stabilization process and maintain their biological functions.

EXAMPLE 12 This example shows that DNA/RNA can be incorporated inside and on the surface of protein spheres before stabilization with a stabilizing agent.

A commercial preparation of calf thymus DNA was added to HSA to provide a concentration of 5 Ag of DNA per mL.

Control spheres were formed with HSA without DNA. Ethanol was added to produce turbidity. The spheres thus formed were stabilized by incubating the mixtures overnight with 5 ML of sodium bisulfite (0.1 M) per mL suspension.

The spheres were then washed in normal saline (3 x 10 mL) and digested in trypsin to release any DNA previously incorporated inside or on the surface of. spheres. Detection of DNA was performed by the Intvogen DNA DipStick Kit, which showed strong positivity with the trypsin digest of spheres o prepared from DNA-containing HSA solutions, but not from control spheres or the supernatants of both experimental and 20 control sphere suspensions.

The experiment was repeated with RNA where all the reagents were also added a RNAase inhibitor. The findings were similar to DNA spheres.

These experiments showed that the spheres can be used as stable carriers for gene therapy for either in vitro or in vivo experiments and therapy.

EXAMPLE 13 This examples illustrates that DNA and RNA can bind to o spheres during the stabilizing phase.

The experimental protocol of Example 12 was followed except that protein particles were first formed by addition of ethanol to 1.0 mL of HSA in the absence of DNA or RNA (with RNAase inhibitor). Within 0.5 min of addition of the stabilizing agent (sodium bisulfite 0.1 5 ML of a suspension of DNA or RNA was added to the protein spheres to result in a final concentration of 5 Mg DNA/RNA per mL of protein suspension.

After digestion of the spheres with trypsin, the protein solution was checked for the presence of liberated DNA or RNA.

Only spheres which had DNA or RNA added at the stabilization phase showed strong positivity. Control spheres or the supernatant of the last wash from either the experimental spheres or control spheres gave negative results when tested for the presence of DNA or RNA.

EXAMPLE 14 Additional agents representative of different chemical groups were used to determine their ability to stabilize spheres formed by adding 70% ethanol to a 15% HSA solution. microliters of a 1M or 1mM solution of the potential stabilizing agent was added to 2 mL of sphere suspensions.

Only two final concentrations (10mM and 10 micromolar) of the agents in the sphere suspensions were studied initially for screening purposes. After two hours of incubation, 5 mL of normal saline was added to 2 mL of sphere suspension. A change from turbidity back to a clear solution indicated that the spheres were not stabilized by either concentration of the agents used. If turbidity remained after 30 minutes, a wider concentration of effective agents was added to sphere suspensions to determine the minimal final concentration that could stabilize the spheres. The agents were as follows: Anions: 20 ammonium chloride ammonium nitrate ammonium sulfate ammonium phosphate, monobasic and dibasic potassium iodide 25 potassium acetate potassium bicarbonate molybdic acid, sodium salt Cations: cobalt chloride cupric chloiride magnesium chloride manganese chloride ferric nitrate Vitamins: cyanobalamin

(B

12 ergocalcifero

(D

2 Acids: folinic acid 6-aminolevulinic acid boric acid cholic acid Amino acids: D,L-phenylalanine poly-L-lysine, MW 114,700 and MW 430,500 Carbohydrates: 2-amino-2-deoxy--D-galactopyranose Antibiotics: penicillin, sodium salt gentamycin chioramphenicol N-acetyl compounds: N-acetylneuraminic acid w-acetamido-2-deoxy-D-glucose Amines: ethanolamine others: choline bitartrae cephalothin glycerol heparin.

chear germ agglutinin (lectin) D-sorbitol laminin dimethyl sulfoxide Results: All of the agents except the-following were ineffective in stabilizing spheres either at the final concentration of 10 mM or 10 micromolar: Effective Chemicals minimal Final Concentration

(MM)

magnesium~ chloride 5 mM manganese chloride 0.5 mM -y-aminolevulinic acid 0.002 mM *gentamycin 0. 05 ing/mL poly-L-lysine, MW 147,000 0.1 mg/mL poly-L-lysine, MW 430,500 0.1 mg/mL N-acetylneuraminic acid 0.5 mg/mL dimethyl suif oxide 1/100 dilution Samples of suspensions were examined under the microscope and found to consist of ionodispersed spheres with no aggregates seen.

EXAMPLE Interaction of Na Bisulfite with 15% HSA before Adding Alcohol to Form Spheres I. Rationale Previous examples show that Na bisulfite stabilizes the microspheres. In this study, the HSA solution was preincubated with Na bisulfite for different time spans before adding alcohol, and examines the effect on the stability of the resulting spheres.

II. Procedure 1. Incubate 1 mL of 15% HSA with 25 pL of 0.1 or 0.05M Na bisulfite for 5, 10, 20, 30, 60, and 120 minutes, respectively, before adding 70% ethanol to form spheres.

2. 1 and 60 minutes after forming spheres, add 3x volume (7.5mL) of normal saline and shake well.

3. Record the time needed to dissolve the spheres.

III. Results The times needed to redissolve the spheres was as follows.

Adding normal saline 1 minute Adding normal saline 60 minutes after Incubation after forming spheres forming spheres Incubation Time 0.05M 0.1M NaHS0 3 0.05M NaHSO 3 0.1M NaHSO 3 (minutes) NaHSO, immediately immediately 2 hours more than 8 hours immediately immediately 2.5 hours more than 8 hours immediately immediately 2.5 hours more than 8 hours 30 immediately immediately 2.5 hours more than 8 hours immediately immediately 2.5 hours more than 8 hours 120 immediately immediately 2 hours more than 8 hours Control immediately immediately (no NaHSO 4 r IV. Discussion 1. The data show that pre-incubation of Na bisulfite in a solution of HSA for up to two hours did not improve the stability of the spheres if challenged within one minute of formation of spheres. In contrast, the incubation time of the spheres with Na bisulfite made a difference.

2. One minute of interaction of this agent with spheres is not sufficient for stabilization. However, if normal saline was added after 2 hours of incubation, the spheres were stable against resolubilization.

3. A final concentration of 2.4 millimolar Na bisulfite is more effective than a final concentration of 1.2 millimolar in promoting the stability of the spheres.

EXAMPLE 16 Pre-mixing Na Bisulfite with Ethanol Before Making Spheres Purpose To study the possibility of premixing Na bisulfite with ethanol before making the spheres and to study the stability of these spheres.

II. Procedure 1. Make 1, 0.5, 0.25, 0.1, 0.05, 0.025, 0.0125, 0.00625, 0.003125, 0.0015625, 0.00078125, and 0.000390625M Na bisulfite solutions in 70% ethanol.

2. Add the Na bisulfite containing alcohol solutions to 1 mL of 15% HSA to make spheres. Monitor the amount of alcohol solution needed.

3. Incubate for 2 hours.

4. Check the appearance of the sphere suspensions grossly and microscopically.

30 5. Add 3x volume of normal saline, mix well, and check the stability of the spheres.

III. Result 1. The Na bisulfite did not dissolve in 70% ethanol well.

The highest concentration obtainable was 0.05M solution.

2. The amount of alcohol needed to make spheres remained the same as with pure 70% ethanol, regardless of the concentration of Na bisulfite. It took about 1.5mL of alcohol/Na bisulfite solution per 1 mL of 15% HSA to make spheres.

3. Appearance after incubating for 2 hours was as follows: 10 n i NaHSO 4 Concentration After (AM) Appearance After 2 Hours Adding In Normal In Ethanol Suspension Gross Microscopic Saline 50,000 30,000 gelatinous aggregates precipitat ion 25,000 15,000 gelatinous aggregates precipitat ion 12,500 7,500 gelatinous aggregates precipitat ion 6,250 3,750 turbid 5+ aggregates turbid 4+ 3,125 1,875 turbid 5+ aggregates turbid 4+ 1,562 937 turbid 5+ aggregates turbid 4+ 781 468 turbid 5+ aggregates turbid 3+ 390 234 turbid 5+ aggregates turbid 2+ 195 117 turbid 4+ no turbid 2+ aggregate 98 58 turbid 4+ no turbid 1+ aggregate 48 29 turbid 3+ no turbid 1+ aggregate 24 14 turbid 2+ no dissolved aggregate 0 0 turbid 3+ individual dissolved spheres IV. Discussion These results show that the spheres can be stabilized by premixing the ethanol with the reducing agent, Na bisulfite. The optimal concentrations are between approximately 29 and 117 micromolars in the suspension.

EXAMPLE 17 Ability of Spheres Stabilized by Na Bisulfite to Bind Fibrinogen This example shows that protein molecules other than albumin fibrinogen) can be bound on the surface of the spheres in the presence of a stabilizing agent and that there is an optimal time for the addition of these surface molecules after the formation of the spheres.

Occasionally it is necessary to bind large quantities of certain biological molecules (such as fibrinogen) on the surface of spheres (to serve as platelet substitutes), but such biological molecules may have limited solubility in its preparative medium. Therefore, a large volume of the biological solution (at the highest possible concentration) will have to be mixed with the spheres soon after their formation. Mixing in a large volume of such non-alcohol containing solution to a partically stabilized sphere suspension may redissolve some of the spheres. However, if the biologically active molecule solution is added after the spheres were completely stabilized by the stabilizing agent may 20 result in unstable or insufficient binding of the biologically active molecule on a per sphere basis. This experiment demonstrates the effectiveness of binding fibrinogen using three different methods of stabilizing the spheres.

I. Procedure 25 1. Make spheres by adding Na bisulfite in one of the following methods: a. to'15 HSA before adding 70% ethanol; b. to 70% ethanol before adding 15% HSA; or c. within 1 minute after forming the spheres by 30 adding 70% ethanol to 15% HSA, 2. The final Na bisulfite concentration in all three methods will be about ImM.

3. Add 0.5x volume of 0.5mg/mL fibrinogen 10, 30, 60, and 120 minutes after the spheres are formed per 1.0 volume of sphere suspension.

4. Compare the amount of .fibrinogen bound per billion spheres, by using a special ELISA method for measuring fibrinogen content.

II. Result Concentration of Spheres (x 10-9/mL of Time of Adding Suspension) Prepared by Adding NaHSO 4 Fibrinogen After Forming (minutes) in HSA in Ethanol Spheres 10 (All spheres dissolved after adding fibrinogen.) (Part of the spheres dissolved after adding fibrinogen. All spheres then dissolved during the washing process.) 4.399 4.164 2.485 4.277 4.175 2.388 120 .3.945 5.842 3.474 3.876 5.802 3.474 Mean Diameter (gm) of Spheres Prepared by Time of Adding Adding NaHSO 4 Fibrinogen After Forming (minutes) in HSA in Ethanol Spheres 1.018 1.078 1.191 1.018 1.081 1.195 120 1.278 1.260 1.193 1.282 1.261 1.193 Fibrinogen Bound (mg/l x 10 9 Spheres) in Time of Adding Spheres Prepared by Adding NaHSO 4 Time of Adding Fibrinogen After Forming (minutes) in HSA in Ethanol Spheres 0.078 0.080 0.106 120 0.085 0.023 0.116

I

IV. Discussion 1. The spheres had to be incubated in the stabilizing agent for more than 30 minutes to stabilize the spheres.

2. All three different methods of adding stabilizing agent could bind fibrinogen onto the spheres.

3. When the stabilizing agent was premixed in HSA before adding alcohol to form spheres, the timing of adding fibrinogen had no effect on the amount of fibrinogen bound.

4. When the stabilizing agent was pre-mixed in the alcohol before mixing with HSA solution to form spheres, the ability of the spheres to bind fibrinogen diminished with time.

When the stabilizing agent was added after the spheres were formed, the timing of adding fibrinogen had no effect on the amount of fibrinogen binded per sphere.

6. In terms of fibrinogen/sphere, adding the stabilizing agent after the spheres were formed resulted in more fibrinogen bound per billion spheres than the other two methods.

For albumin-based particles, additional substances serving a therapeutic or diagnostic function, or -both, can be entrapped 20 within the particle bulk and carried by the particles to a site for in vivo administration. Examples of classes of such substances are enzymes, amino acids, peptides, nucleic acids, contrast agents and nonmacromolecular therapeutic drugs. A contrast agent of particular interest is technetium. The 25 incorporation of these additional substances is conveniently achieved by combining them with the protein solutions from which the proteins are precipitated to form the particles.

Additional substances can also be attached non-covalently to the exterior surfaces of the particles. Examples of substances which are useful when adhered to the particles in this manner are proteins, immunoglobulins and nucleic acids, as well as molecular species in general which exhibit specific binding to biological molecules such as cell surface receptors.

Specific examples of particular interest are fibrinogen and peptides which contain reactive sequences of fibrinogen, such as aspartine-glycine-aspartic acid (RGD). Adherence of these substances to the particle surfaces is conveniently achieved by contacting the substances with the particles soon after the particles are formed.

Electron microscopy of platelet aggregates formed by activation of platelets by ADP in the presence of fibrinogen coated particles clearly shows co-aggregation of such particles within the platelet mesh. In contrast, similar particles without fibrinogen coated on the surface do not get entrapped into any platelet aggregates. There are four possible mechanisms of action of fibrinogen-coated particles in coaggregation with platelets or in improving bleeding time.

These are as follows: When platelets become active by contact with a wound site, hidden receptors become exposed and bind fibrinogen molecules through the RGD site. When a fibrinogen molecule bound to particulate matter is captured by an activated platelet, the particle is also entrapped by the clot.

Fibrinogen on the surface of platelets is further converted into fibrin by the action of thrombin.

After thrombin releases the Fibrinopeptide A and B from fibrinogen, a thrombin-binding site would be exposed on the remainder of the fibrinogen molecule. It was important that thrombin be kept locally, not only to enhance the cascade of clotting factors in the vicinity of *e the wound, but more importantly to prevent the formation 20 of thrombosis downstream or DIC (disseminated intravascular coagulation) which can be fatal. The presence of fibrinogen coated synthetic particles in the locality of a wound may have provided more surface to bind thrombin and may thus have kept the clotting mechanism locally near the wound.

f Addition of thrombin to a concentrated solution of fibrinogen would result in the formation of a soft clot. Such a clot (without platelets) will not form in sub-minimal concentrations of fibrinogen. On the other 30 hand, addition of thrombin to a concentrated suspension of fibrinogen-coated particles alone also fails to aggregate the particles. This is probably due to the long distance between fibrin on the surface of one particle and the fibrin on the surface of a neighboring particle, so that no polymerization is possible, However, it was found that a moderate concentration of fibrinogen-coated particles mixed in a sub-minimal concentration of soluble fibrinogen would readily form a clot after the addition of thrombin.

It is therefore apparent that the soluble fibrinogen when acted upon by thrombin would form a bridge between the particles to link them all up as a clot. Fibrinogen is present in the plasma typically between 200 to 300 mg/L.

Therefore, at a site, such as a wound site, where thrombin is present, a fibrin clot will more readily form in the presence of fibrinogen coated particles than without them.

It was also found that upon activation, platelets release ATP and serotonin. In a "thrombocytopenic" suboptimal) concentration of platelets, the rate of serotonin release is also suboptimal. Mixing fibrinogen-coated particles with_ platelets in the absence of activating agents does ndt lead to release of serotonin. However, activation of a suboptimal concentration of platelets in the presence of fibrinogen-coated particles leads to an improved rate of serotonin release. It is therefore conceivable that natural platelets respond to the rate with which they come into contact with other fibrinogen-containing bodies, which would result in an improved rate of release of factor and the recruitment of other platelets.

It has also been found that heparin-treated animals have improved bleeding time when fibrinogen-coated particles are injected, as compared to infusion of normal saline or control spheres without fibrinogen. Rheologically, it is expected that the small size of such particles would cause them to be close to the endothelial cells instead of being in the middle of the blood vessel. This may be one reason why small particles coated with fibrinogen can be effective in control of bleeding, 25 because they are more concentrated where needed. Fibrinogencoated particles are also effective in patients who have platelet dysfunction, due for example to aspirin ingestion, or in animals treated with neuraminidase, or in renal dysfunction patients.

Additional experiments showed that the material with which the core or matrix of the particles was formed was not significant to the clotting process as long as a sufficient amount of fibrinogen was bound either covalently or noncovalently to the surface of the particles.

35 In addition to proteins, other substances are useful in the formation of the core or matrix with which or on which fibrinogen can bind or coat. These include lipids, nucleic acids, biopolymers such as polytactic acid, and different classes of polysaccharides. The following list is illustrative but not limiting: A variety of biocompatible matrices can be conjugated to fibrinogen to provide particles suitable for treating thrombocytopenia, reducing bleeding time and blood loss, ameliorating platelet dysfunction due to kidney failure, drug sensitivity, drug action aspirin, or antiplatelet antibodies) or as a result of cardiopulmonary bypass. They are particularly useful in treating patients who have developed resistance to platelet transfusion. The term "biocompatible" is employed in its conventional sense, that is, to denote compounds which do not substantially interfere or interact with the tissues, fluids and other components of the body in an adverse fashion in the:particular application of interest.

These matrices include microspheres made from natural and synthetic polymers, phospholipid vesicles (see U.S. Patent No.

4,904,479 and 4,728,578), polyesters and/or polyamides encapsulated in methylcellulose Patent No. 5,233,995), gelatin, albumin, collagen Patent No. 4,844,882), aliphatic and alicyclic carbon-containing compounds (U.S.

Patent No. 5,143,716), polyaminoacids Patent No.

4,247,406), polyglutamate, and pblylysine either separately or in combination with proteinaceous microspheres albumin EP 0 633 030, WO 92/17213, WO 91/12823 synthetic polymers (WO 92/17514, U.S. Patent Nos. 4,089,800, 4,572,869, 3,429,827, polyesters, polymers and copolymers polylactide/polyglycolide and polylactones ecaprolactone Patent Nos. 4,637,605, WO 92/18164) polysilicones 2,026,513B), cyclodextrins (GB 2,090,738B, WO 93/02712, WO 92/21382) non-proteinaceous cross-linked or 25 polymerized amphophilic moieties (WO 92/17212), hydrophilic synthetic polymers such as polyalkylene glycols PEG), polyvinylpyrrolidones Patent 5,470,911).

EXAMPLE 18 Thrombospheres (TS) are cross-linked human serum 30 albumin spheres (mean diameter 1.2 um) with human fibrinogen covalently bound on the surface. The present studies were done to evaluate the effect of TS on Bleeding Time Blood Loss (BL) and on platelet survival following infusion of TS.

Similar results are obtained with other fibrinogen coated 35 particles (crosslinked or non-crosslinked), particularly albumin particles, where the fibrinogen is adsorbed noncovalently onto the surface.

Methods: Severely thrombocytopenic rabbits (av plt count <10x10 3 /ul) received either TS, 7.5x10 9 /kg; control albumin spheres (CS) which had no fibrinogen, 7.5x10 9 /kg; or an equal volume of normal saline Ear BT were done 1, 24, 48, 72 hours after treatment and expressed as the.mean ±SD for each group of rabbits in seconds. BT measurements were stopped if BT of an animal exceeded 900. BL (in ml) was measured from the radioactivity in the collection vessel from blood lost from an ear wound 24 hour after treatment in rabbits previously infused with 1 ml of Cr-51 labeled erythrocytes. Survival of Cr-51 labeled platelets (in hr) in TS treated normal rabbits were also measured.

Results n BT(lh) BT(24h) BT(48h) BT(72h) TS 46 401±171 351±116 334±154 547±265 CS 10 570±58 882±58 895±16 >900 NS 22 898±12 >800 888±43 >900 The infusion of TS was associated with a significantly lower BL than seen in NS-treated rabbits (1.1 vs.

5.1 ml), while platelet survival was normal in both TS and NS treated normal rabbits.

Conclusions: The data indicate that TS shorten the BT in severely S 20 thrombocytopenic rabbits for up to 72 hours and significantly reduce blood loss. The normal platelet survival time indicates that TS is a safe hemostatic agent without thrombogenicity.

Without being bound by any particular theory, it is believed that in areas away from the wound without 25 thrombin because it has been rapidly removed by antithrombin or the liver), fibrinogen on the surface of the particles remains undigested and therefore unreactive. However, at the wound site, a region having thrombin and activated platelets those with some digested fibrinogen (fibrin) on their surface), S 30 the locally trapped thrombin will digest the fibrinogen on the particle surface, thus activating the thrombosphere to participate in clot formation. Since all patients have some platelets, albeit at low levels, and such platelets are activated only at the wound site, the thrombospheres only enhance clotting activity at wound sites and not elsewhere. In essence, the thrombospheres behave as agents which enhance and amplify clotting action at a wound site. Since a minimal level of activated platelets are needed, thrombospheres enhance clotting only where needed and do not have the adverse effects associated with clot formation elsewhere. Thus, thrombospheres are of particular value in treating diseases where clotting is slow due to low concentrations of activated platelets, e.g., thrombocytopenia.

The following list of biocompatible matrices is illustrative but not limiting: cellulose agarose hemicellulose starch (amylose, amylopectin) mannans glucans xanthans pullutans arabinans arabinogalactans arabinoglucans arbinoglucoronomannans xylans arabinoxylans carrageenans colominic acid glycosaminoglycans (for example:. heparin, heparin sulfate, dermatan sulfate, chondroitin sulfate, keratan sulfate, hyaluronans) The binding of fibrinogen to the particles may occur by 25 one or both of the following mechanisms: In the case of human serum albumin as the matrix material, the spheres may be stabilized by the addition of crosslinking agents. It is possible that one reactive site of the crosslinking agent such as glutaraldehyde is 30 covalently bound to the sphere while the other reactive site is bound to the fibrinogen. However, when fibrinogen is added to the spheres in the presence of a low concentration of cross-linking agent, in the presence of 10,000 molar excess of a competing small molecule (such as glycine, for example, competing for the aldehyde site), fibrinogen can still bind to the surface of the sphere.

In fact, transmission electron microscopy (cross section) shows a thick layer of fibrinogen (verified by immunochemical means to be fibrinogen) which is far more than one molecular layer thick, surrounding the surface and filling inside the interior of the protein sphere.

Such experiments suggest that hydrophobic bonding, may be responsible.

Hydrophobic bonding: in the presence of a suitable agent (such as alcohol), the hydrophobic sites of the core component molecules would be exposed. This allows the molecules to stack up to not only form a particle (and thereby desolvate from solution), but also to provide a surface suitable for fibrinogen to attach to, either covalently (via cross-linking after stacking onto the surface of the particle, or before, or at the tite the fibrinogen is attached to the surface of interior of the particle) or noncovalently.

As used herein, the terms "treatment" or "treating" of a condition and/or a disease in a mammal, means: preventing the condition or disease, that is, avoiding any clinical symptoms of the disease; (ii) inhibiting the condition or disease, that is, arresting the development or progression of clinical symptoms; and/or (iii) relieving the condition or disease, that is, causing the regression of clinical symptoms.

20 The conditions and diseases treated in the present invention include thrombocytopenia, reducing bleeding time and blood loss, ameliorating platelet dysfunction due to kidney e failure, drug sensitivity, drug action aspirin, or antiplatelet antibodies) or as a result of cardiopulmonary 25 bypass. Treating patients who have developed resistance to platelet transfusion is of particular value. In general, any platelet related disease, whether caused by low platelet levels or platelet dysfunction despite platelet levels being normal, are treatable by the methods and compositions disclosed herein.

As used herein, the term "therapeutically effective amount" refers to that amount of a biocompatible matrix containing a bioactive substance which, when administered to a mammal in need thereof, is sufficient to effect treatment (as defined above).

The compounds of this invention are administered at a "therapeutically effective dosage, that amount which, when administered to a mammal in need thereof, is sufficient to effect treatment, as described above. Administration of the active compounds and salts described herein can be via any of the accepted modes of administration for agents that serve similar utilities.

The level of the drug in a formulation can vary within the full range employed by those skilled in the art, from about 0.01 percent weight to about 99.99%w of the drug based on the total formulation and about .01%w to 99.99%w excipient. Preferably the drug is present at a level of about to about Generally, an acceptable daily dose is of about 0.001 to mg per kilogram body weight of the recipient per day, preferably about 0.05 to 25 mg per kilogram body weight per day, and most preferably about 0.01 to 10 mg per kilogram body weight per day. Thus, for administration to a 70 kg person, the dosage range would be about 0.07 mg to 3.5 g per day, preferably about 3.5mg to 1.75 g per day, and most preferably about 0.7 mg to 0.7 g per day depending upon the individuals and disease state being treated. Such use optimization is well within the ambit of those of ordinary skill in the art.

Administration can be via any accepted systemic or local route, for example, via parenteral, oral (particularly for infant formulations), intravenous, nasal, bronchial inhalation aerosol formulation), transdermal or topical routes, in the form of solid, semi-solid or liquid dosage forms.

Actual methods of preparing such dosage forms are known, or will be apparent, to those skilled in this art; for example, see Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pennsylvania, 16th Ed., 1980. The composition to be administered will, in any event, contain a quantity of 25 the active compound(s) in a pharmaceutically effective amount for relief of the particular condition being treated in accordance with the teachings of this invention.

The following is a.list of examples of specific substances which can be incorporated into the composition of particles of the present invention.

1. Protein A 2. Concanavalin A 3. IgG 4. hemagglutinin 5. transferrin 6. von-Willebrand factor 7. anti-human factor IX monoclonal antibody 8. anti-CD8 monoclonal antibody 9. goat anti-clathrin 10. fibronectin 11. human fibroblast growth factor-acidic, recombinant 12. human interleukin-2, recombinant 13. anti-human platelet-derived growth factor beta receptor 14. anti-beta-lipoprotein alpha 2-macroglobulin 16. streptokinase 17. anti-progesterone antibody 18. anti-leukotriene B4 antibody 19.

CGGRGDF-NH

2 doxorubicin 21. daunarubicin 22. EDTA-conjugated to HSA 23. DTPA-conjugated to HSA 24. technetium gadolinium 26. HSA conjugated to FITC (Fluorescein Isothiocyanate) 27. HSA conjugated to TRITC (Tetramethylrhodamine B isothiocyanate) 28. HSA conjugated to PE (Phycoerythrin) 29. HSA conjugated to Ferritin HSA conjugated to biotin 31. alkaline phosphatase 32. peroxidase 33. amphotericin B 25 34. Adjuvant peptide (N-acetylmuramyl-1-alanyl-d-isoglutamine) HIV-1 protease substrate (acetyl-ser-gln-asn-tyr-pro-val-val-amide) 36. Fe30 4 magnetite or magnetic particles 30 37. cysteine-cyclohexanol conjugate 38. HIV-glycoprotein 120 39. anti-CD4 antibody 40. fibrinogen Albumin-based particles containing Tc99m either in their bulk or on their surfaces are illustrative of the use of these particles as vehicles for specific agents. The incorporation or attachment of Tc99m can be achieved through direct covalent bonding or through a chelating agent. Examples of chelating agents are cysteine-cyclohexanol conjugate and

DTPA.

The chelating agent may be pre-bonded to soluble HSA molecules which are then mixed with other HSA molecules during the formation of the original aqueous protein solution.

Alternatively, chelating agents may be covalently bonded directly to preformed particles. A third alternative is to add the chelating agent as one of the biological molecules, not covalently bound to any HSA molecules. The chelating agent will then be trapped within the particles or near their surfaces when the particles are formed.

The procedure of binding the Tc99m to the particles, with or without chelating agents, can follow standard nuclear medicine procedures. For example, stannous chloride or other reducing agents (0.01 to 0.3 mg) can be added to approximately 1 mg of particles suspended in a suitable buffer to reduce the sulhydryl groups in the protein molecules. Sodium pertechnetate Tc99m (5 to 250 millicurie) is then added to the suspension. The excess reducing agent reduces the pertechnetate (Tc04-) to TcO 2 which then binds to the sulhydryl group on the protein molecules, or to the sites on the chelating agents designed to bind the Tc0 2 It is expected that more Tc99m binds to particles through chelating agents than without chelating agents.

Alternatively, stannous chloride and lyophilized particles could be stored as a dry powder in the absence of oxidizing agents, to be reconstituted as a suspension by the addition of Tc99m solutions.

The presence of chelating agents has the additional advantage of possibly stabilizing the TcO0 2 before it binds to 25 the protein molecules.

An alternative method would be to allow pertechnetate Tc99m to be reduced by a reducing agent in the presence of a free chelating agent, one which is not yet associated oo ~with the particles, then binding the Tc99m-chelating agent 30 conjugate to the particles.

The particles may alternatively be reduced by a different reducing agent after which they can be purified and stored as a reduced dry (lyophilized) powder, while the pertechnetate would be reduced by a different kind of reducing agent immediately before interaction with the already reduced particles. Due to the short half life of Tc99m, the product which results from the mixing of the pertechnetate-containing liquid with the particle suspension or powder should be ready for injection into a patient within much less than one hour and without the need for other purification. Examples of reducing agents are dithiothreitol, dithioerythritol, ascorbic acid, 2-mercaptoethanol, and pyrophosphate. In addition, the reduced TcO 2 may first be stabilized by an intermediate product involving D-glucarate.

A wide variety of bioactive molecules can be incorporated within the interior, on the surface, or near the surface of the particles. Combinations of one or more compounds can also be incorporated into a single particle.

Examples of biologically active molecules that can be incorporated include, but are not limited to: drugs, biologically active peptides, polypeptides, carbohydrates, lipids, lipoproteins, glycoproteins,. enzymes, ligands, i receptors, radioactive compounds, fluorescent or excitable compounds, imaging materials, oxygen-carrying materials, toxins, anti-toxins, neurologically active materials, chemotherapeutic agents, chelating compounds, nucleotides, nucleoside, nucleic acids, polynucleotides, antibiotics, magnetic materials, and nutrients. Further examples are subunits or fragments of these molecules, as well as analogs of the molecules, their competitors, inhibitors, and antagonists, antibodies against them, antibodies against antibodies against them, receptors to which they will bind, anti-sense entities (whether in the form of RNA, DNA or even protein forms), and the genes from whose information they are derived.

The following are examples of biologically active molecules which can be incorporated in the manner described above.

Lipids: methanoic, ethanoic, propanoic, butanoic, pentanoic, hexanoic, heptanoic, octanoic, nonanoic, decanoic, undecanoic, dodecanoic, tridecanoic, tetradecanoic, pentadecanoic, hexadecanoic, heptadecanoic, octadecanoic, nonadecanoic, eicosanoic, heneicosanoic, docosanoic, tricosanoic, tetracosanoic, pentacosanoic, hexacosanoic, heptacosanoic, octacosanoic, nonacosanoic, triacontanoic, hentriacontanoic, 10-undecenoic, cis-9-tetradecenoic, trans-9-tetradecenoic, cis-9-hexadecenoic, trans-9-hexadecenoic, cis-10-heptadecenoic, cis-6-octadecenoic, trans-6-octadecenoic, cis-7-octadecenoic, cis-9-octadecenoic, trans-9-octadecenoic, cis-l-octadecenoic, trans-ll-octadenenoic, cis-12-octadecenoic, cis-13-octadecenoic, cis-12-hydroxy-9-octadecenoic, trans-12-hydroxy- 9-octadecenoic, cis-9,12-octadecadienoic, trans- 9,12-octadecadienoic, 9,11(10,12)-octadecadienoic, cis-6,9,12-octadecatrienoic, cis-9,12,15-octadecatrienoic, cis-6,9,12,15-octadecatetraenoic, cis-8-!eicosenoic, cis-11-reicosenoic, cis-13-eicosenoic, cis-li, l4eicosadienoic, 8, 11-eicosatrienoic, 5' 8, 1l-eicosatriynoic, cis-8, 11,l4eicosatrienoic, cis-li, 14, 17-eicosatrienoic, cis-5,8,ll,14-eicosatetraenoic, 5, 8,11,l4eicosatetraynoic, 8,11, 14, 17-eicosapentaenoic, cis-13-docosenoic, trans-l3docosenoic, cis-13 ,16-docosadienoic, cis-13,16,19-docosatrienoic, cis-7,1O,13,16docosatetraenoic, cis-4, 7,10,13,16, 19-docosahexaenoic, cis-iS-tetracosenoic acids.

Lectins: Abrus precatorius (Agglutinin, Abrin A toxin, Abrin C toxin), Agaricus bisporus, Anguilla anguilla, Arachis hypogaea, Bandeiraea simplicifofla (BS-I, BS-I-B4, BS- I-AB3, BS-I-A2B2, BS-I-A3B, BS-I-A4, BS-II), Bauhinia purpurea, Caragana arborescens, Cicer arietinun, Codium fragile, Concanavalin A, SuccinylConcanavalin A, Datura stramonium, Dolichos biflorus, Erythrina corallodendron, Erythrin a cristagalli, Euonymus europaeus, Glycine max, Helix aspersa, Helix pomatia, Lathyrus odoratus, Lens culinaris, Limulus polyphemus (Bacterial agglutinin), Lycopersicon esculentun, Maclura pomifera, Momordica charantia, Mycoplasma gailisepticum, Naja mocambique mocambique, Naja naja kaouthia, Perseau americana, 25 Phaseolus coccineus, Phaseolus limensis, Phaseolus vulgaris (PHA-E, PHA-L), Phytolacca americana, Pisum.

sativum, Pseudmonas aeruginosa PA-I, Psophocarpus tetragonolobus, Ptilota plumosa, Ricinus communis (Toxin, RCA6O, Toxin, RCA12O), Robinia pseudoacacia, Sambucus nigra, Solanum tuberosum, Sophora japonica, Tetragonolobus purpureas, Triticum vulgaris, Ulex europaeus (UEAI, UEAII), Vicia faba, Vicia sativa, Vicia villosa (A4, B4), Vigna radiata, Viscum album,-Wisteria floribunda.

Complement proteins, either as single proteins or as 35 combinations of several proteins: Cig, C2, C3, C4, C5, C6, C7, C8, C9, Properdin factor B.

0 0 Spin labels and spin traps: Doxyl Nitroxides, Proxyl Nitroxides, e.g., 3- (4-nitrophenoxycarbonyl) -proxyl; Tempo Nitroxides, Tempo; DL-t-Butyl Nitroxide; Spin traps: Nitrosobenzene, Nitrosadisulfonic acid, 2 -methyl-2 -Nitroso-Propane.

Arachidonic -acid cascade and related compounds: HETEs: 5 -HETE C5 -hydroxy-6-trans-8-cis-1I1-cis-14 -ciseicosatetraenoic acid]; 11(S) -HETEll cis-8-cis-12-trans-14 -cis-eicosatetraenoic acid] 12 -HETE (12 -hydroxy-5-cis-8-cis-1o-trans- 14-cis-eicosatetraenoic acid) ;12(S) -HETE(12 hydroxy-5-cis-8-cis-1O-trans- 14-cis-eicosatetrae-noic acid]; 15(S)-HETE(15(S)-hydroxy-5-cis-8-cis-11--Cis- 13-trans-eicosatetraenoic acid]; HPETEs: 5(S) -HPETE -hydroperoxy-6-trans-8-cis-1-cis- 14-cis-eicosatetraenoic acid]; 12(S) -HPETE(12 hydroperoxy-5-cis-8-cis-1 0-trans- 14 -ciseicosatetraenoic acid); 15(S) -HPETE hydroperoxy-5-cis-8-cis-1 1-cis-13 -transeicosatetraenoic acid]; DiHETEs: 5(S) ,6(R)-DiHETE(5(S) -dihydroxy-7-trans-9trans-11-cis-14-cis-eicosatetraenoic acid]; ,12(S)-DiHETE(5(S) ,12 (S)-dihydroxy-6-trans-8-cislotrans-14-cis-eicosatetraenoic acid]; 5(S) DiHETE(5(S) ,15(S)-dihydroxy--6-trans-8-cis-ll-cis-13trans-eicosatetraenoic acid] Other arachidonic acid cascade related compounds: 13-Azaprostanoic acid; Baicalein; 7-7dimethyleicosadienoic acid; 5,8, lleicosatriynoic acid; 25 5,8,11,14-eicosatetraynoic acid; oleoyloxyethyl Phosphocholine; sodium furegrelate; w-3 fatty acids; leukotrienes (LTA4, LTB4, LTC4, LTD4, LTE4); lipoxin (A4, B4), Prostaglandins (A2, B2, D2, El, E2, F2a, 12, G2, H12); 16-16-Dimethyl-prostaglandin E2; 6-Keto-prostaglandin Fla; 2, 3-Dinor 6-keto-prostaglandin Fla~; 9, l1-Dideoxy-9a, 11amethanoepoxyprostaglandin-F2ac; Carbacycl1in; Thromboxanes (CTA2, B2, A2); p-Arbutin; H-Arg-gly-Asp-OH; H-Arg-Gly- Asp-Ser-Pro-Ala-Ser-Ser-Lys-Pro-OH; Ascorbate oxidase; ascorbic acid; asparagine; aspartic acid; arachidonic acid Ion channel modulators: :Amiloride, Baicalein, BAY K 8644, Bepridil, Brevetoxi~n a. (PbTx-l, PbTx-2, PbTx3, PbTx-7, PbTx-9), w-Conotoxin GVIA, Conus geographus, Diltiazem, Methoxyverapamil, Nifedipine, Ryanodine, 9,21, -Dehydro-Ryanodine, Saxitoxin, Tetrodotoxin, TMB-8, Toxin II, Verapamil.

Biologically active peptides: 4- (2-Acetamido-2-deoxy-beta-D-Glucopyranosyl) -N-acetylmura myl-L-Ala-D-Glu Amide N-Acetyl-Asp-Glu N'-Acetyl-Cholecystokinin and its f ragments N-Acetyl-Hirudin and its fragments Acetyl -Leu-Leu-Arginina 1 N-Acetyl -Leu-Leu-Meth joninal N-Acetyl-Leu-Leu-Norleucinal Acetyl-Met-Asp-Arg-VaI.-Leu-Ser-Arg-Tyr N-Acetyl -Met-Leu-Phe N-Acetylmuramyl-D-a lanyl-D- isoglutamine N-Acetylmuramyl -L-a lanyl-D-isoglutamine N-Acetylmuramyl-L-alanyl-L-isoglutamine N-Acetylmuramyl-Ala--D-isoglutaminy 1-Ne-stearoyl-Lys N-Acetyl-Phe-Nle-Arg-Phe Amide Acetyl-Renin Substrate Tetradecapeptide Acetyl-Ser-Asp-Lys-Pro Acetyl-Ser-Gln-Asn-Tyr Acetyl-Ser-Gln-Asn-Tyr-Pro-Val-Val Amide Acetyl-Ser-Tyr-Ser-Met-Glu-Hi s-Phe-Arg-Trp-G ly-Lys-Pro-Val Amide N-Acetyl-Thr-I le-Nie-Phe (CH 2 NH) -Nle-Gln-Arg Amide ACTH (Adrenocorticotropic Hormone) Adrenal Cyclase Activating Polypeptide-27 Adrenal Medulla Peptides Adrenal Peptide E we Adrenocorticotropic Hormone and fragments 25 Adrenorphin Adipokinetic Hormone II Adjuvant Peptide Ala-Arg-Pro-Gly-Tyr-Leu-Ala-Phe-Pro-Arg-Met Amide beta-Ala-Arg-Ser-Ala-Pro-Thr-Pro-Met-Ser-Pro-Tyr Ala-D-gammna-Glu-Lys-D-Ala-D-Ala Ala-Gly-Cvs-Lys-Asn-Phe-Phe-Trp-Lys-Thr-Phe-Thr-Ser-Cvs Ala-Gly-Ser-Glu Ala-D-isoglutaminyl-Lys-D-Ala-D-Ala Ala-Leu-Ala-Leu Ala-Leu-Ile-Leu-Thr-Leu-Val-Ser Ala-Lys-Pro-Ser-Tyr-Hyp-Hyp-Thr-Tyr-Lys beta-Ala-Ser-His-Leu-G ly-Leu-Ala-Arg Ala-Ser-Thr-Thr-Thr-Asn-Tyr-Thr D-Ala-Ser-Thr-Thr-Thr-Asn-Tyr-Thr Amide Aldosterone Secretion Inhibiting Factor Allatotropin Alytes in Amastatin beta-Anyloid and fragments Angiogenin and fragments Angiotensin I and analogs Angiotensin II and analogs Angiotensin III and analogs Angiotensin Converting Enzyme Inhibitor Angiotensinogen and fragments Angiotonin Anorexogenic Peptide Anthrani lyl-His-Lys-Ala-Arg-Val-Leu-p-Nitro-Phe-G lu-Ala-Ni e-Ser Amide Antide Anti-inflammatory Peptide 1 Antipa in Antireproductive Tripeptide Ap am in Arg-Arg-Leu-I le-Glu-Asp-Ala-G lu-Tyr-Ala-Al a-Arg-G ly Arg-Arg-Leu-I le-Glu-Asp-Asn-Glu-Tyr-Thr-Al a-Arg-Gly Arg-Arg-Lys-Ala-Ser-Gly-Pro Arg-Gly-Asp Arg-Gly-Asp-Ser Arg-Gly-Asp-Ser-Pro-Ala-Ser-Ser-Lys-Pro Arg-Gly-Glu-Ser Arg-G ly-Phe-Phe-Tyr-Thr-Pro-Lys-Ala Arg-Gly-Pro-Phe-Pro-Ile Arg-His-Phe Arg-Lys-Arg -Ala-Arg-Lys-G lu Arg-Lys-Asp-Va 1-Tyr Arg-Lys-Glu-Va 1-Tyr o3 Arg-Phe-Asp-Ser Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met Amide Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg Arg-Ser-Arg-His-Phe Arg-Tyr-Leu-Pro-Thr Arg-Tyr-Val1-Val-Leu-Pro-Arg-Pro-Val-Cys-Phe-Glu-Lys-Gly-4e t-Asn-Tyr-Thr-Va l-Arg' Asn-Al a-G ly-Ala Asn-Pro-Asn-Ala-Asn-Pro-Asn-Ala Asn-Pro-Asn-Ala-Asn-Pro-Asn-Ala-Asn-Pro-Asn-Ala Asp-Ala-Glu-Asn-Leu-I le-Asp-Ser-Phe-Gln-Glu-I le-Val Asp-Arg-Va 1-Tyr-i le-His-Pro-Phe-H is-Leu Asp-Leu-trp-Gin-Lys Asp-Ser-Asp-Pro-Arg Atrial Natriuretic Peptide and fragments Atriopeptins Auriculin Avidin Beauvericin Bestatin Biocytin-Neuropeptide Y Biotin/biotinylated peptides N-t-BOC-beta -Ala-Trp-Met-Asp-Phe Amide N-t-BOC-beta-Ala-Trp-Met-beta- (Ben zyl) Asp-Phe Amide N-t-BOC-Gly-Trp-Met-Asp-Phe Amide N-t-BOC-Gly-Trp-Met-beta- (benzyl) Asp-Phe Amide N-t-BOC-Glu-Glu-Ile Methyl Ester N-t-BOC-Glu-Glu-Leu Methyl Ester N-t-BOC-Glu-Glu-Val Methyl Ester N-t-BOC-Lys-Pro-Tyr-Ile-Leu Methyl Ester N-t-BOC-Met-Asp-Phe Amide N-t-BDC.-Met-Leu-Phe N-t-BOC-Nle-Leu-Phe N-t-BOC-Phe-D-Leu-Phe N-t-BOC-Phe-Leu-Phe-Leu-Phe N-t-BOC-Trp-Asp-Phe Amide N-t-BOC-Trp-Met-Asp-Phe Amide N-t-BOC-Trp-Met-Phe Amide Bombesin and analogs 25 Bradykinin and analogs Bradykinin Potentiator 5a, 9a, B, C) Brain Natriuretic Peptide, Brefeldin A Buccalin 30 Bursin Caerulein Calcitronin, Cactoi GeeReaedPptd beaCalcitonin Gene Related Peptide Calcitonin Gene Related Peptide fragment 8-37 Calcitonin Precursor Peptide *:Calmodulin-Dependent Protein Kinase II (fragment 290-309) Calpain Inhibitor I Calpain Inhibitor II Calpain Inhibitor Peptide Carassin N-Carboxymethyl-Phe-Leu N- R, S] -2-Carboxy-3-phenylpropionyl) -L-Leucine Cardicexcitatory Peptide aipha-Casein and fragments Beta-Casomorphin Na-CBZ-Arg-Arg-Pro-Phe-His-Sta-I le-His-Ne-BOC-Lys Methyl Ester CBZ-Leu-Val-Gly Diazomethyl Ketone N-CBZ-D-Phe-Phe-Gly N-CBZ-Pro-D-Leu7 N-CBZ-Pro-Leu-Gly Hydroxamate CD4 and fragments Cecropins Cerebellin Chemostactic Peptides Cholecystokinin and fragments Chorionic Gonadotropin and fragments Chymostatin Circumsporozoite (CS) Protein of Plasmodium falciparum repetitive sequences Collagen Conotoxin GI p-conotoxin CluIB w-conotoxin GVIA a-conotoxin SI Copper Binding Peptide 25 Corazonin Corticotropin A oCorticotropin-Like Intermediate*Lobe Peptide Corticotropin Releasing Factor and analogs Tyr-Corticotropin Releasing Factor ,QCorticotropin Releasing Factor Antagonist C-Peptide and fragments Cyclic-AMP Dependent Protein Kinase Substrate Cyclo (7-Arninoheptanoy l-Phe-D-Trp-Lys-Thr [Hz1)) Cyclo (D-Asp-Pro-D-Val-Leu-D-Trp) Cyclohexylacetyl-Phe-Arg-ser-Val-G ln Anide Cyclo (His-Phe) Cyclo (His-Pro) Cyclo (Leu-Gly) Cyclo (Phe-Ser) Cyclo(Pro-Gly) 3 Cyclo (D-Trp-Lys-Thr-Phe-Pro-Phe) Cyclo (D-Trp-Lys-Thr-Phe-Pro-Tyr) Cys-Gin-Asp-Ser-Glu-Thr-Arg-Thr-Phe-Tyr Cys-Ser-Arg-Ala-Arg-Lys-Gln-Ala-Ala-Ser-I le-Lys-Val-Ala-Va l-Ser-Ala-Asp-Arg Cys-Tvr-Ile-Gln-Asn-Cvs-Pro-Arg-Gly Amide Cys-Tvr-Ile-Gin-Asn-Cvs-Pro-Leu Gly Amide Cvs-Tvr-I le-Ser-Asn-Cys-Pro-I le-Gly Amide DAGO .(D-Ala2, Dalargin Decorin Delta Sleep Inducing Peptide Dermenkephalin Dermorphin Diabetes Associated Peptide Amide Diazepam Binding Inhibitor and fragments Diprotin A Diprotin B DNA Binding Peptide Dynorphin and fragments Echistatin Elastatinal Elastin Chemotactic protein and fragment Eledoisin Eledoisin-Related Peptide.

*Endothel1in aipha-Endorphin 25 beta-Endorphin and fragments gamma-Endorphin Endothel1ins Enkephalin, Leucine and analogs Enkephalinamide, Leucine and analogs '0 Enkephalin, Methionine and analogs Enkephalinamide, Methionine and analogs Enzyme Inhibitors Eosinophi lotactic Tetrapeptides Epiamastatin Epibestatin Epidermal Growth Factor Epidermial Mitosis Inhibiting Pentapeptide Experimental Allergic Encephalogenic Peptide Erythropoietin. fragmen t 1-26 Fibrinogen-Binding Inhibitor Peptide Fibrinogen Related Peptide Fibrinogen A and analogs Fibrinogen B and analogs Fibroblast Growth Factor, Acidic fragment 1-11 Fibroblast Growth Factor, Basic fragment 1-24 Fibronectin-Binding Protein Peptide D3 Fibronectin fragments and analogs Fibronectin Related Peptide Fibronectin Pepsin Fibronectin Chymotrypsin 40K, 45K, 120K) Fibronectin Trypsin 30K, 60 K) N-FMOC-val-Gly-Gly--t-Butyl-Tyr-Gly-O-t-Butyl-Tyr-Gly-Ala -Ne-CBZ-Lys N-Formyl-Met-Leu-Phe Formyl-Peptides Foroxymithine FTS (Serum Thymic Factor) Galanin Message Associated Peptide and fragments Galanin Gastric Inhibitory Polypeptide Gastrin-I and fragments Gastrin I, Big Gastrin II Pentagastrin Gastrin Releasing Peptide Castrin-Tetrapeptide Amide Gastrointestinal Peptides Gi lodeliquescin 25 Gin-Ala-Thr-Val-Gly-Asp-Ile-Asn-Thr-G lu-Arg-Pro-Gly-Met-Le Gln-AJa-Thr-Val-Gly-Asp-Va l-Asn-Thr-Asp-Arg-Pro-Gly-Leu-Le u-Asp-Leu-Lys Gln-Arg-Arg-Gln--Arg-Lys-Ser-Arg-Arg-Thr-l le JO Gln-Lys-Arg-Pro-Ser-G ln-Arg-Ser-Lys-Tyr-Leu Glu-Ala-Glu Glu-Ala-Glu-Asn G lucagon Glucagon(l-37) Glucagon-Like Peptide I and fragments Glu-Leu-Ala-Gly-Ala-Pro-Pro-Glu-Pro-Ala Glutathione and analogs G ly-Arg-Ala-Asp-Ser-Pro Gly-Arg-Ada-Asp-Ser-Pro-Lys Gly-Arg-Gly-Asp Gly-Arg-G ly-Asp-Asn-Pro Gly-Arg-Gly-Asp-Ser Gly-Arg-Gly-Asp-Ser-Pro Gly-Arg-Gly-Asp-Ser-Pro-Lys Gly-Arg-Gly-Asp-Thr-Pro Gly-Arg-Gly-Leu-Ser-Leu-Ser-Arg Gly-Arg-Tyr-Asp-Ser Gly-Gin Gly-Glu-Gln-Arg-Lys-Asp-Va 1-Tyr-Va l-Gin-Leu-Tyr-Leu Gly-Gly-Arg Gly-Gly-His Gly-Gly-Tyr-Arg Gly-His-Arg-Pro Gly-His-Lys Gly-Leu-Met Amide Gly-Pen-Gly-Arg-Gly-Asp-Ser-Pro-Cys-Ala (Pen2, Cys9 cyclic) Gly-Phe-Asp-Leu-Asn-Gly-Gly-Gly-Va 1-Gly Gly-Pro-Arg Gly-Pro-Arg-Pro Gly-Pro-Gly:-Gly Granuliberin R Granulocyte Macrophage-Colony Stimulating Factor GRF 1-40, Human Growth Hormone Release Inhibiting Factor Growth Hormone Releasing Factor and fragments H-142 Hepatitis A viral'proteins and peptides 25 Hepatitis B Virus Pre-S Region (120-145) Hepatitis C Viral proteins and peptides Herpes Virus Ribonucleotide Reductase Inhibitors Heterotypic Adhesion Receptor Hirudin and fragments His-Asp-Met-Asn-Lys-Va l-Leu-Asp-Leu His-Leu-Gly-Leu-Ala-Arg His-Lys-Ala-Arg-Va l-Leu-p-Nitro-Phe-G lu-Ala-Nle-Ser Ainide V.00D-His-Pro-Phe-His-Leu-ph-

[CH

2 -Leu-Va 1-Tyr Histones HIV Envelope Protein (gp4l) fragment 579-601 *HIV Envelope Protein (gpl2O) fragments :HIV Protease Inhibitor HIV Substrate, III HIV viral protein and peptides Histone H2A fragment 86-120 Hydra Peptide and fragments Hypercalcemia Malignancy Factor Hypertrehalosaemic Neuropeptide Iberiotoxin Ile-Pro-Ile le-Va l-Pro-Phe-Leu-Giy-Pro-Leu-Leu-G ly Leu-Leu-Thr Amide Immunostimulating Peptides Inhibin, alpha subunit, fragment 1-32 Insulin Chain A, oxidized Insulin Chain B, oxidized Insulin Chain B fragment 22-30 Insulin Chain C Insulin-Like Growth Factor I Insulin-Like Growth Factor II Integrin alpha 4, alpha V beta 5 alpha2, alpha3, alpha 5, alpha V, beta 1, beta 2, beta 4) Interleukin lB fragment (163-171) Interleukin-2 Receptor C-Terminal Sequence Interleukin 1 alpha, 2, 6, gamma) Isotocin Kallidin Kallikrein Inhibitor Kass inin Katacalcin and analogs Kemptide and analogs Kentsil Kinetensin and analogs 25 Laminin and fragments (929-933) Leu-Arg-Arg-Ala-Ser-Leu-Gly go Leu-Arg-Arg-Ala-Hse-Leu-Gly Leu-Arg-Arg-Trp-Ser-Leu-Gly too. Leucokinins Leucopyrokinin and fragments Leu-Leu Methyl Ester Leu-Lys-Lys-Phe-Asn-Ala-Arg-Arg-Lys-Leu-Lys-Gly-Ala-I le-Le u-Thr-Met-Leu-Ala Leu-Pro-Pro-Ser-Arg Leu-Ser- (pN402) -Phe-Nle-Ala-Leu Methyl Ester Leupept in LH-Rl (Luteinizing Hormone Releasing Hormone) and analogs too.:beta-Lipotropin and fragments Litorin Lys-Arg-Pro-Pro-G ly-Phe-Ser-Pro-Phe-Arg Lys-Ala-Arg-Va l-Nle-p-Nitro-Phe-Glu-Ala-Nle Amide Lys-Arg-Thr-Leu-Arg-Arg Lys-Cys-Thr-Cys-Cys-Ala Lys-Glu-Glu-Ala-Glu Lys-His-Gly Amide Lys-Lys-Arg-Ala-Ala-Arg-Ala-Thr-Ser-Amide Lys-Lys-Asp-Ser-Gly-Pro-Tyr Lys-Lys-Gly-Glu Lys-Phe-lle-Gly-Leu-Met Amide Lys-Pro-Pro-Thr-Pro-P-Pro.Glu-Pro.G lu-Thr Lys-Trp-Lys D-Lys-Tyr-D-Trp-D-Trp-Phe D-Magainin II Amide Magainin I Magainin II Manning Compound Manning-Binding Protein Mast Cell Degranulating Peptide Mast Cell Degranulating Peptide HRI Mast Cell Degranulating Peptide HR2 Mastoparan Aiphal-Mating Factor MCD Peptide Aipha-Melanocyte Stimulating Hormone and analogs Beta-Melanocyte Stimulating Hormone Delta-Melanocyte Stimulating Hormone Melittin Merosin Met-Asn-Tyr-Leu-Aa-Phe-Pro-Arg-Met Amide Met-Gln-Met-Lys-Lys-Val-Leu-Asp-Ser Met-Gly-Trp-Asn-Ser-Thr-Thr-Phe-His-GinThr.Leu-Gln.Asp-Pr o-Arg-Va l-Arg-Gly-Leu-Tyr-Phe-Pro-Ala-Gly-Gly Met-Leu-Phe Metorphamide Molluscan Cardioexcitatory Peptide Morphiceptin Morphine Modulating Neuropeptide Morphine Tolerance Peptide Motilin

MSH

Muramyl Dipeptides Beta-Naphthyl-D-Ala-C sTrDTpLsvics.Thr Amnide coendorphin Beta-Neoendorphin Alpha-Neurokinin Beta-Ala8)-alpha-Neurokinin fragment 4-10 Neurokinin A, Nle-10, B, MePhe7-B) Neurozuedins B,C) Neuropeptide K Neuropeptide Y Neurotensin and analogs N-Nicotinoyl-Tyr- (Nalpha-CBZ-Arg) -Lys-His-Pro-I le Nle-Arg-Phe Amide Nle-Sta-AJla-Sta NeutrAvidin Octadecaneuropeptide 6, 7, 8) Osteocalcin fragment 7-19 Osteocalcin fragment 45-49 Oxyntomodul in Oxytocin and analogs PACAP27 Amide Pancreastatin and fragments Pancreatic .Polypeptide Parathyroid Hormone and fragments Pardaxin Pentagastrin 20 Pepstatin A Peptide II of T, wagleri venom Peptide T Peptide YY pGlu-Ala-Glu 25 pGlu-Ala-Lys-Ser-Glu-Gly-Gly-Ser-Asn pGlu-Asn-Gly pGlu-Asp-Pro-Phe-Leu-Arg-Phe Amide pGlu-Gln-Arg-Leu-Gly-A2sn-Gln-Trp-AJa-Val-Gly-His-Leu-Met Amnide (SO3H) -Thr-Gly-Trp-Met-Asp-Phe Amide pGlu-Glu-Asp-Ser-Gly pG lu-Gly-Leu-Pro-Pro-Arg-Pro-Lys-Ile-Pro-Pro pGlu-Gly-Leu-Pro-Pro-Gly-Pro-Pro-I le-Pro-Pro pGlu-His-Gly pGlu-His-Gly Amide pGlu-His-Pro pGlu-His-Pro Amide pGlu-His-Pro-Gly pGlu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly Amide pGlu-Leu-Tyr-Glu-Asn-Lys-Pro-Arg-Arg-Pro-Tyr-Ile-Leu pGlu-Lys-Arg-Pro-Ser-Gln-Arg-Ser-Lys-Tyr-Leu pGlu-3-Methyl-His-Pro Amide (pGlu4)-Myelin Basic Protein 4-14 pGlu-Ser-Leu-Arg-Trp Amide pGlu-Thr-Ser-Phe-Thr-Pro-Arg-Leu Amide: pGlu-Trp-Pro-Arg-Pro-Gln-l1le-Pro-Pro pGlu-Val-Asn-Phe-Ser-Pro-Gly-Trp-Gly-Thr Amide Paracelsin Peptide 6a (D-Alal]-peptide T amide Phe-Gly-Gly-Phe Phe-Gly-Leu-Met Amide Phe-Gly-Phe-Gly Phe-Leu-Arg--Phe Amide Phe-Leu-Glu-Glu-I le Phe-Leu-Glu-Glu-Leu Phe-Leu-Glu-Glu-Va 1 Phe-Leu-Phe-Gln-Pro-Gln-Arg-Phe Amide Phe-Met-Arg-Phe Amide Phe-Met-Arg-D-Phe Amide Phe-Met-rD-Arg-Phe Anude Phe-D-Met-Arg-Phe Amide D-Phe-Met-Arg-Phe Amide Phe-Ser-Trp--Gly-Ala-Glu-Gly-Gln-Arg-

PHI

Phosphoramidon Phosphate Acceptor Peptides (Val6, Ala7) -Kemptide 25 Physalaemin *Platelet Derived Growth Factor (AB-chain, heterodimer, AA homodimer, BB homodimer) Platelet Membrane Glycoprotein IIB Peptide Releasing Hormone fragment 14-26 Pressinoic Acid N-proCalcitonin 1-57 Proctol1in Prodynorphin 228-240 Proenkephal1in Pro-His-Pro-Phe-His-Phe-Phe-Val-Tyr-Lys Pro-Leu-Gly Amide Pro-Phe-Gly-Lys Pro-Leu-Ser-Arg-Thr-Leu-Ser-Val-A1 a-Ala-Lys-Lys Prosomatostatin 1-32

PYY

Protected Marine Adhesive Peptide Protein A Protein G (binds to Fc region, specially of IgGl subclass) Protein Kinase C Protein Kinase C Substrate Protein Kinase Inhibitor Pro-Thr-Pro-Ser Amide

PTH

PTH-Related Protein (1-40) Ranatens in Renin Inhibitors Renin Substrate Tetradecapeptide N- (alpha-Rhamnopyranosyloxyhydroxyphosphinyl) -Leu-Trp Sarafotoxin Schizophrenia Related Peptide Secretin Senktide Ser-Asp-Gly-Arg-G ly Ser-Gln-Asn-Phe-Phi (CH 2 N) -Pro-Ile-Val-Gin Ser-Gin-Asn-Tyr-Pro-I le-Val Ser-Ile-rGly-Ser-Leu-Ala-Lys Ser-Phe-Leu-Leu-Arg-Asn-Pro-Asn-Asp-Lys-Tyr-Glu-Pro-Phe Serglycin Serum Thymic Factor and analogs Sexual Agglutination Peptide Sleep Inducing Peptide Small Cardioactive Peptide B Somatostatin and analogs 25 Speract Streptavidin Streptolysin Substance P and analogs Tumor Antigen C-Terminal Sequence 0D Syndyphalin Syntide Tenascin Terlipressin Thapsigargin DL-Thiorphan Thr-Lys-Pro-Arg Thrombin Receptor Activator Thr-Phe-G ln-Ala-Tyr-Pro-Leu-Arg-Glu-Ala Thr-Pro-Arg-Lys Thr-Ser-Lys Thr-Thr-Tyr-Ala-Asp-Phe-I le-Ala-Ser-G ly-Arg-Thr-G ly-A-rg- Arg-Asn-Ala-Ile-His-Asp Thr-Tyr-Ser Thr-Val-Leu Thrombospondin Thymopoetin fragments Thymos in Thymosin fragments Thyrocalcitonin Thyrotropin Releasing Hormone and Related Peptides Tocinoic Acid Toxin, Snake Transforming Growth Fa ctor-Alpha

TRH

Trp-Ala-Gly-Gly-Asp-Ala-Ser-Gly-Glu D-Trp-Ala-Trp-D-Phe Amide Trp-His-Trp-Leu-Gln-Leu Trp-His-Trp-Leu-Gln-Leu-Lys-Pro-Gly-Gln-Pro-Met-Tyr Trp-His-Trp-Leu-Ser-Phe-Ser-Lys-Gly-Glu-Pro-Met-Tyr Trp-Het-Asp-Phe Amnide Trp-Nle-Arg-Phe Amide Tuftsin and analogs Tumor Necrosis factor alpha) Tyr-Ala-Gly-Ala-Val- Val1-Asn-Asp-Leu Tyr-D-Ala-Phe-Asp-Val-Val-Gly Amiide Tyr-D-Ala-Phe-Glu-Val--Val-Gly Amide Tyr-Gly-Ala-Va 1-Va l-Asn-Asp-Leu 25 Tyrosine Protein Kinase Substrate Tyr-D--Ala-Gly Tyr-Gly-Gly V Tyr-D-Ala-Phe-Met Amide 30 Tyr-Gly-Gly Tyr-Gly-Gly-Phe-Leu Tyr-Gly-Gly-Phe-Leu-Arg-Arg-I le-Arg-Pro-Arg-Leu-Arg-Gly-- Kminopentylamide Tyr-Gly-Gly-Phe--Met-Arg-Arg-Val Tyr-Gly-Gly-Phe-Met-Arg-Arg-Va1 Amide Tyr-G ly-Gly-Phe-Met-Arg-G ly-Leu Tyr-Gly-D-Trp-Phe-D-Phe Amide Tyr-I le-Gly-Ser-Arg Tyr-Phe-Met-Arg-Phe Amide Tyr-Pro-Leu-Gly Amide Tyr-Pro-Phe-Pro Amide Tyr-Pro-Val-Pro Amide Tyr!-D-Ser-G ly-Phe-Leu-Thr Tyr-D--Trp-Ala-Trp-D-Phe Amide 00: Tyr-D-Trp-Ala-Trp-D-Phe Methyl Amnide Urodilatin Urotensin I Urotensin II Val-Ala-Ala-Phe Val-Arg-Lys-Arg-Thr-Leu-Arg-Arg-Leu Va l-Glu-Glu-Ala-Glu Val-Glu-Pro-I le-Pro-Tyr Val-Glu-Ser-Ser-Lys Val-Gly-Asp-Glu Val-Gly-Ser-Glu Val-Gly-Val-Ala-Pro-Gly Va 1-His-Leu-Thr-Pro Va l-His-Leu-Thr-Pro-Val -Glu-Lys Val-Ile-His-Asn Valosin Va l-Pro-Leu Vasoactive Intestinal contractor Vasoactive Intestinal Peptide and analogs Vasopressin and analogs Vasotocin Versican Vitronectin Xenopsin Yeast Alpha-Factor Human serum albumin glycoconjugates: Carboxyethylthioethyl 2-Acetaiido-2-deoxy-3-0-Beta-Dgal actopyranosy l-beta-D-glucopyranos ide-HSA Conjugate Carboxyethylthioethyl 2-Acetamido-2-deoxy-4-Q-beta-D- 0 galactopyranosyl-beta-D-glucopyranos ide-HSA Conjugate Carboxyethylthioethyl 2 -Acetaxnido-4 (2 -acetamido-2 deoxy-beta-D- glucopyranosyl-6-0- (alpha-Lfucopyranosyl) -2-deoxy-beta-D--glucopyranoside-HSA Conjugate Carboxyethylthioethyl galactopyranoside-HSA Conjugate Carboxyethylthioethyl 4-0- Glucopyranosyl-alpha-D-glucopyranosy 1) -alpha-Dglucopyranosyl) -beta-D-glucopyranoside-HSA Conjugate Free oligosaccharides and simple derivatives: 2 -Acetamido- 6-0- (2 -acetamido-2-deoxy-beta-Dglucopyranosyl) -2-deoxy-D-glucopyranose 2 -Acetamido-2-deoxy- 3-0-beta-D-ga lactopyranosyl-Dga lactopyranose 2 -Acetamido-2 -deoxy-4 (4 -0-Beta-D-galatopyraliosyl-beta- D-galactopyranosyl) -D-glucopyranose 2-Acetamido-2-deoxy-3 -0-beta-D-galactopyranosyl-Dglucopyranose 2 -Acetamido-2 -deoxy- 6-0-beta-D-ga lactopyrariosyl-Dglucopy-ranose* (2-Acetamido-2 -deoxy-4 (beta-D-galactopyranosy-1) beta-D-glucopyranosyl) -D-ga lactopyranose (2 -Acetamido-2 -deoxy-beta-D-glucopyranosyl) -Dgalactopyranose (2 -Acetamido-2 -deoxy-beta-D-glucopyranosyl) -beta- D-galactopyranosyl) -D-glucopyranose N-Acetyl lactosamine Benzyl 2 -Acetamido-6-0- (2 -acetamido-2 -deoxy-beta-Dglucopyranosyl) -2 -deoxy-alpha-D-glucopyranos ide Benzyl 2 -Acetamido-2 -deoxy-3 -0-beta-D-galactopyranosylalpha-D-galactopyranoside Benzyl 2 -Acetamido-2 -deoxy-3 -0-beta-D-galactopyranosyl bet a-D-glucopyranoside 20 Benzyl n-Butyl 4-0-beta-D-G alactopyranosyl-beta-D-glucopyranoside D- -Cellobiose D- -Cellopentaose D- -Cellotetraose D-(+)-Cellotriose Digalacturonic acid Ethyl 2-Acetamido-2-deoxy-4-0- (4 -0-a lpha-Dgalactopyranosyl-beta-D-galactopyranosyl) -beta-Dglucopyranos ide Ethyl 2 '-Fucosyllactose 3-Fucosyllactose 4 -0-alpha-D-Galactopyranosyl-D-galactopyranose lactopyranose 4-0- (4-0-beta-D-Galactopyranosyl-beta-D-galactopyranosyl) D-G lucopyranose 4 -0-beta-D-Galactopyranosyl-D-mannopyranose glucopyranosyl) alpha-D-glucopyranosyl) -Dglucopyranose Lacto-N-tetraose 3 -0-alpha-D-Mannopyranosyl-D-mannopyranose Methyl 4-0- (2 -Acetainido-2-deoxy-4-0-beta-Dgalactopyranosyl-beta-D-glucopyranosyl) -beta-Dgalactopyranosyl) -beta-D-glucopyranoside Methyl 3-0- (2-Acetamido-2-deoxy-beta-D-glucopyranosyl) beta-D-galactopyranoside Methyl 6-0- (2-Acetamido-2-deoxy-beta-D-glucopyranosyl) a lpha-D-mannopyranos ide Methyl 3, 6-Di-0- (alpha-D-mannopyranosyl) -alpha-Dmannopyranos ide Methyl 3 -0-beta-D-Ga lactopyranosy 1-beta -Dgalactopyranoside (2 -0-Methyl-beta-D-galactopyranosy) -D-glucopyranose Methyl 4 -0-beta.-D-Galactopyranosyl-beta-D-glucopyranoside Methyl Methyl Methyl 4-0-a lpha-D-Mannopyranosy 1-alpha-D-mannopyranos ide Methyl 6-0-alpha-D-Mannopyranosyl-a lpha-D-mannopyranoside n-Propyl 4-Q-beta-D-Galactopyranosyl-beta-Dglucopyranos ide 20 Trigalacturonic acid Activated oligosacchar ides: Carboxnethoxyethylthioethyl 2-Acetamnido-2-deoxy-4-0-beta-Dgalactopyranosyl-beta-D-glucopyranos ide Carbomethoxyethylthioethyl 4-0- Glucopyranosyl-alpha-D-glucopyranosyl) -alpha-Dglucopyranosyl) -beta-D-glucopyranoside p-Nitrophenyl 2-Acetamido-2-deoxy-3-0- (2-acetamido-2deoxy-bcta-D-glucopyranosyl) -alpha-Dgalactopyranos ide itrophenyl 2 -Acetamido-2-deoxy-3 (Beta-Dga lactopyranosyl) -alpha-D-ga lactopyranos ide p-Nitrophenyl 2-Acetamido-2-deoxy-3-0 -beta-Dgalactopyranosyl-beta-D-glucopyranos ide p-Nitrophenyl galactopyranoside p-Nitrophenyl alpha-D-Maltopentaoside Neo-glycolipids: octadecylthioethyl ga lactopyranoside Octadecylthioethyl 4-0- alpha-D-glucopyranosyl) -alpha-D-glucopyranosyl) -beta- D-glucopyranoside Coagulation proteins and various factors, and their fragments, inhibitors, receptors to which they bind, or genes and information molecules from which they may be derived: Acutase; Agkistrodon contortrix Thrombin-like Enzyme; Ancrod; a2-Antiplasmin; Antithrombin III; Atroxin; Coagulation Factors; Coagulation factor Inhibitors; Crotalase; Ecarin; Factor I, II, III, IV, V; Factor V Activating Enzyme; Factor VI, VII; VIII; Von-Willebrand Factor; Factor IX; Factor X; Factor X, Activiated (Xa) Factor X Activating Enzyme; Factor XI, XII, XIII; Fibrin; Fibrin/Fibrinogen Degradation Products; Fibrinogen; Fibrinolytic proteins; Heparin; Hirudin; Kallikrein; Plasmin; Plasminogen; Plasminogen Lysine Binding Sites; Platelet Factor 4; Platelet Aggregation Reagent; Brain Cephalin; Snake venoms; Streptokinase; Thrombins; Thromboplastin; Thromboplastin with Calcium; Tissue Plasminogen Activator; Urokinase Agents and drugs used to treat or prevent HIV infection: Alpha interferon, Interleukin-2, Amphotericin B, Amphotericin B Methyl Est; Ampligen (polyl-polyC; C12U); AS-101 (ammonium trichloro(dioxyethylene-O-O' tellurate); CD8+ lymphycyte proteins; HIV viral proteins, cell receptor anagonists, cell receptor binding proteins; azidothymidine and analogs or conjugates; Azidouridine (including analogs or conjugates); beta interferon; Carbovir; Carrisyn; Colchicine; Colony Stimulating Factors; Compound Q; D4T (2',3'-didehydro-3'-dideoxythymidine); DTC (Imuthiol); Dextran Sulfate; Dideoxycytidine; Dideoxyinosine; DHEA (dehydroepiandro-sterone); Doxorubicin; gamma globulin; HIV-immunogen; Hypericin; tyrosine-glycine dipeptide; tyrosine-glycine-glycine tripeptide; Isoprinosine; Lentinan (beta-(l-3)-glucan); Lipid compounds, e.g. AL-721 or EL-721 and like products; Peptide T; Polio Vaccine proteins; soluble CD4; CD4-linked toxins; Ribavirin; SMS 201-995 (a long-acting analog of somatostatin); Thymic Humoral Factor; Thymopentin; Tumor Necrosis Factor; ketoconazole; fluconazole; Eflornithine; Spiramycin; Ganciclovir (DHPG); Foscarnet; Acyclovir; Vibaradine; Pyrimethamine; sulfadiazine; TMP/SMX; Amikacin; Ansamycin; Ciprofloxacin; Clofazamine; Cycloserine; Imipenum; Ethambutol; Isoniazide; Rifampin; Streptomycin; sulfa based antibiotics; pentamidine; Dapsone/trimethoprim; steroids; Trimetrexate with Leukovorin; Clindamycin; primaguin; Dapasone; Spiramycin; piritrexim Adjuvant pe-ptides: N-acetylmuramyl-L-alanyl-D-isoglutamine N-acetylmuramyl-D-alanyl-D-isoglutaiine N-acetylmuramyl-L-alanyl-L-isoglutamine N-acetylmuramyl-L-a 1.anyl -D-isoglutamine- N-acetyl-glucosaminyl-beta -N-acetylmurainyl-L-alanyl- D-isoglutamine Components of Freund's Complete Adjuvant Components of Freund's Incomplete Adjuvant Dimethyldioctyldecyl Amumonium Bromide Lipoproteins and related enzymes: Apolipoprotein A (I and II), B, CIII, CII, CI, E High Density Lipoprotein Low Density Lipoprotein Very Low Density Lipoprotein Lipoprotein Lipase Lipoteichoic Acid Lipoxidase Diaphorase 0:00 Lipoxin A4, B4 Lipoxygenase Prostaglandan Synthetase chelating agents: Iminodiacetic Acid dimethyl-ida, paraisopropyl-ida,parabutyl-ida, diisopropyl-ida, diethyl-idA) V0600EDTA (Ethylenediaminetetraacetic acid) 0 NTA (Nitriloacetic acid) TPP (Tripolyphosphate Cysteime DEDTC (Diethyldithiocarbamate) citric acid Tartaric acid Penicillamine

EGTA

Caged calcium chelators: NITR7, DM-nitrophen, NITrRS/AM; Ammonium Nnitrosophenyl-hydroxylamine; Amimonium purpurate; alpha-Benzoin oxime; N, N-Bis-(hydroxyethyl)-glycine; 2, 3-butane-dione dioxime; Trans-l, 2-Diaminocyclohexanetetra-acetic acid (CDTA); Diethylenetriaminopenta-acetic acid (DTPA); 4, benzene-l, 3-disulphonic acid; 2, 3-Dimercapto-l- Propanol; Diphenylthio-carbazone; Dipyridyl; 3, 6-Disulpho-1, 8-dihydroxy-naphthalene; Dithiooxamide; Eriochrome Black T; Ethylene-diamine; Ethylenediaminetetraacetic acid (EDTA); (Ethylenedioxy) -diethylenedinitrilo-tetraacetic acid (EGTA); o-Hydroxybenzaldehyde oxime; N- (2 Hydroxyethyl) iminodiacetic acid (HINDA); 8-Hydroxyquinoline; 8.-Hydroxygjuinoline-5-sulphonic acid;- 4- Methyl-i, 2-dimercapto-benzene; Nitrilotriacetic acid (NTA); 5-Nitro-l,10-phenanthroline;. 1,10- Phenanthroline; Potassium ethyl xanthate; Salicylic acid.; sodium diethyldithio-carbamate; 2-Thenoyl-2furoylmethane; Thenoyl-trifluoro-acetone; Thiourea; Triethylenetetramine Deferoxamine mesylate Edetate Calcium disodium meso 2,3-dimercapto succinic acid Penicillamine Trientine 20 Chelators specially useful in chelating Tc99m: A thiolactone diaminedithiol bifunctional chelating agent p-carboxyethylphenylg lyoxal-di-N-methylthioxemicro-ba zone A diamide dimercaptide chelating agent A hydroxy compound cyclohexanol) attached to 25 cysteine Bisthio semicarbazones Cyclan Diamido dithio ligands Radionuclides: Indium 111 Thallium 201 Technetium 99m Other compounds and chelates suitable for NRI imaging: Gadolinium, cadmium, Strontium, Chromium; ferrous gluconate; manganese; nickel, piperidine and pyrrolidine NSFR derivatives, ferric ammnonium citrate Reagents that can be used to provide spacer arms for bioreactive molecules to extend beyond the immediate surface of the particles: Biocytin Biocytin hydrazide p-Aminobenzoyl Biocytin Enzymes: Alteplase; Anistreplase; Adenosine Deaxninase; Amylase; Angiotensin 1, 11, 111; Calmodulin; Carboxypeptidase; Catalase; Cellulase; Cholesterol oxidase; Cholinesterase; Chymotrypsin; Collagenase; Conplement cascade proteins; Creatine phosphokinase; Deoxyribonuclease I, II; Dipeptidyl peptidase; DNA polymerase; Endoproteinase; Endonucleases; Esterases; beta-Galacatosidase; Galact-ose oxidase; Galactose dehydrogenase; Glucose dehydrogen-ase;.

Glucose oxidase; Glucose-6-phosphate dehydrogenase; Glucuronidase/Aryl sulfatase; Glutamate-oxaloacetate transaminase; Glutamate-pyruvate transaminase; Glutathione reductase; Glutathione peroxidase; Glycopeptidase; Hementin; Hemoglobin; Hexokinase; Hyaluronidase; Lactate dehydrogenase; Lactoperoxidase; Lactamase; Lipase; Myokinase; Neuraxuinidase; Nicotinamide-adenine Dinucleotide kinase; Nicotinamide-adenine Dinucleotide oxidase; Nuclease; Nucleosidase; Papain; Peroxidase; Phenylalanine dehydrogenase; phosphatase (acid or alkaline); Phosphodiesterase; Phospholipase (A2, C, D); .20 Plasmin; Proteases; Protein Kinase C; Proteinase K; Renin; Reverse transcriptase; Ribonuclease T1, T2, U2); RNA polymerase; Sialytransferase; Streptokinase; Subtilisin A; Superoxide dismutase; Terminal transferase; Urease; Urokinase 25 Nucleotides and fragments thereof: anti-sense (DNA or RNA against RNA or DNA, single or double stranded), cloning vectors, coliphage DNA, lambda phage DNA, M13 DNA, Adenovirus DNA, phi-X 174 phage DNA, Simian virus DNA, cytomegalovirus DNA, Epstein-Barr Virus .0 genes, Herpes Simplex genes, ribosomal RNA, human DNA and RNA; Genes coding for ribozymes; genes coding for *.:antibiotics ampicillin, chloramphenicol, cycloserine, gentamycin, kanamycin, kasugamycin, nalidixic acid, rifampicin, spectinomycin, streptomycin, tetracycline) Platelet related proteins and enzymes: Platelet factor 4; 1-3-Dioxolane; l-O-Hexadecyl-2 -acetyl-sn-glucero-3 -phospho- (N,N,N-trimethyl) -hexanolamine; Platelet activating factors 1-0-hexadecyl-2-acetyl-snglycero-3-phosphocholine; 3-O-hexadecyl-2-acetyl-snglycero-1-phosphocholine; l-Q-hexadecyl-sn-glycero-3 phosphocholine; 1-O-hexadecyl-2-N-methylcarbamyl-snglycero-3-phosphocholine; l-O-hexadecyl-2-thioacetyl-2deoxy -sn-glycero-3 -phosphocholirie; 1 -O-hexadecyl-2 -acetylsn-glycero-3-phospho (N-methylpyrrolidino) -ethanolainine); Platelet activating factor 18; 18:1; Lyso-platelet activating factor 18; platelet activating factor-16; Enantic-platelet activating factor-16; Lyso-platelet activating factor-16; trans-2,5-bis- (3,4,5trimethoxyphenyl); 1-0-Hexadecy l-2-acetyl-sn-glycerc-3 phospho N-trimethyl) -hexanolanine.

other compounds: Protein A,B,C,G,S; Ricin A; Proadifen (SKF-525A)l Taxol; Thiolytes; Thiostrepton; Thrombin Thrombocytin; beta-Thromboglobulin; Thrombospondin; Transferrin (apo-, partial iron, holo); Tumor Necrosis factor; Vitronectin, Forskolin, Integrins; caged compounds (caged ATP, caged INsP3, caged cAMP, caged cGMP, caged GTP, caged carbamoyl.

chorine); Mezerein; Plasminogen; Aminocaproic acid; desmopressin acetate; Activase RGD-containing peptides: .Gly-Arg- Gly-Asp-Ser-Pro Gly-Arg-Gly-Asp-Thr-Pro Gly-Arg-Gly-Asp-D-Ser-Pro Gly-D-Arg-Gly-Asp-Ser-Pro G ly-Arg-Gly-Glu-Ser-Pro (inactive control pept ide) -Gly-Arg-Gly-Asp-Asn-Pro n-Methyl-Gly-Arg-Gly-Asp-Ser-Pro Arg-Gly-Asp-Ser Giy-Arg-Gly-Asp-Ser Gly-Arg-Gly-Asp-Ser-Pro-Cys G ly-Arg-Gly-Asp-Ser-Pro-Lys Gly-Arg-Gly-Asp-Ser-Pro-Ala-Ser-Ser-Lys (Gly-Pen-Gly-Arg-Gly-Asp-Ser-Pro-cys-Ala [cyclical]) Antineoplastic agents: 1. Alkylating agents, such as nitrogen mustards (chiorambucil, cyclophosphamide, mechlorethamine, meiphalan), ethyleneamine derivatives (thiotepa), alkyl sulfonates (busuif an), nitrosoureas (carmustine, lomustine), triazenes (dacarbasine) 2. Antimetabolites, such as folic acid analogues (methotrexate), pyrimidine analogues (cytarabine, floxuridine, fluorouracil), purine analogues (mercaptopurine, thioguanine) 3. Natural Products,' such as ymnca alkaloids (vinblastine, vincristine, paclitaxel), podophyllotoxin and its derivatives (etoposide); antibiotics (bleomycin, 63 dactinomycin, doxorubicin, daunomycin, mithramycin, mitomycin) 4. Hormones, such as adrenal corticosteroids (prednisone), estrogens (chlorotrianisene, conjugated estrogens, diethylstilbestrol, diethylstilbestrol diphosphate, ethinyl estradiol), androgens (calusterone, dromostanolone propionate, fluoxymesterone, testolactone, testosterone propionate, testosterone enanthate), progestine (hydroxyprogesterone, medroxyprogesterone, megestrol), antiestorgen (tamoxifen) Enzymes, such as asparaginase 6. Miscellaneous agents, such as substituted urea (hydroxyurea), methyl hydrazine derivative (procarbazine), adrenocortical suppressant (mitotane), heavy metal complex (cisplatin, carboplatin) SThe foregoing is offered primarily for purposes of illustration. It will be readily apparent to those skilled in the art that the materials, proportions, methods of preparation and formulation and other parameters of the various systems described herein may be further modified or substituted in various ways without departing from the spirit and scope of the invention.

Claims (66)

1. A suspension of particles of albumin in a first aqueous medium, said particles being monodisperse in said suspension, and having a size range of from about 5 to about 5000 nanometers, said particles further containing at least one stabilizing compound selected from the group consisting of oxidizing agents, reducing agents, high molecular weight polymers, hydrogen acceptors and sulfur-containing ring compounds in an amount sufficient to prevent said particles from dissolving upon dilution of said suspension with a second to aqueous medium which is alcohol free, to a volume increase of at least about

2. A suspension of particles in accordance with claim 1 wherein said stabilizing agent is a reducing agent, said reducing agent being an organic reducing agent selected from -the group consisting of dithiothreitol, glutathione, and 2- mercaptoethanol.

3. A suspension of particles in accordance with claim 1 wherein said stabilizing agent is a reducing agent, said reducing agent being an inorganic reducing agent selected from to the group consisting of sodium bisulfite, sodium sulfite, stannous chloride, and manganese chloride.

4. A suspension of particles in accordance with claim 1 **wherein said stabilizing agent is an oxidizing agent.

5. A suspension of particles in accordance with claim 1 wherein said stabilizing agent is a high molecular weight polymer selected from the group consisting of polyethylene glycols.

6. A suspension of particles in accordance with claim 1 wherein said stabilizing agent is a hydrogen acceptor selected from the group consisting of NADP and NAD.

7. A suspension of particles in accordance with claim 1 wherein said stabilizing agent is a sulfur-containing ring compound.

8. A suspension of particles in accordance with claim 1 in which said'stabilizing agent is at least about 1% by weight relative to the total of said albumin and said stabilizing agent.

9. A suspension of particles in accordance with claim 1 in which said stabilizing agent is from about 1% to about 30% by weight relative to the total of said albumin and said stabilizing agent.

A suspension of particles in accordance with claim 1 in which said first aqueous medium further includes a lower alkyl alcohol. o1

11. A suspension of particles in accordance with claim 10 in which said lower alkyl alcohol constitutes from about 10% to about 80% by volume of said first aqueous medium.

12. A suspension of particles in accordance with claim 10 in which said lower alkyl alcohol is a member selected from the 15 group consisting of methanol, ethanol, n-propanol, isopropanol and n-butanol.

13. A suspension of particles in accordance with claim 10 in which said lower alkyl alcohol is a member selected from the group consisting of ethanol and n-butanol.

14. A suspension of particles in accordance with claim 1 in which said albumin is human serum albumin.

A suspension of particles in accordance with claim 1, said particles further including biologically active macromolecules other than albumin, said biologically active 15 molecules being immobilized on the exterior of said particles.

16. A suspension of particles in accordance with claim 15 in which said biologically active macromolecules are members selected from the group consisting of proteins, immunoglobulins and nucleic acids.

17. A suspension of particles in accordance with claim 1, in which said particles further comprise a biologically active substance other than albumin, said biologically active substance being distributed substantially homogeneously within said particles.

18. A suspension of particles in accordance with claim 17, in which said biologically active substance is a member selected from the group consisting of enzymes, amino acids, peptides, nucleic acids and nonmacromolecular therapeutic drugs.

19. A suspension of particles in accordance with claim-1, in which said particles further comprise a biologically active substance other than albumin, said biologically active to substance being distributed substantially homogeneously within said particles and immobilized on the exterior of said particles.

A suspension of particles in accordance with claim 1, in which said particles further comprise an imaging agent distributed substantially homogeneously within said particles.

21. A method for the preparation of an aqueous suspension of monodisperse particles of albumin having a size range of from 2o about 50 to about 5000 nanometers in diameter and stable against resolubilization, said method comprising: forming an-aqueous solution containing as solutes: albumin, and (ii) a stabilizing agent selected from the group consisting of oxidizing agents, reducing agents, high molecular weight polymers, hydrogen acceptors, sulfur- containing ring compounds and combinations thereof in a resolubilization suppressing amount; and 2 treating said aqueous solution with a lower alkyl alcohol in an amount sufficient to cause said aqueous solution to become turbid.

22. A method in accordance with claim 21 in which said solutes are used at a concentration such that said particles comprise from about 1.0 to about 150 g per liter of said suspension.

23. A method in accordance with claim 21 in which said 4o alcohol is a member selected from the group consisting of methanol, ethanol, n-propanol, isopropanol and n-butanol. 67

24. A method in accordance with claim 21 in which said alcohol is a member selected from the group consisting of ethanol and n-butanol.

25. A method in accordance with claim 21 in which step (b) comprises adding said lower alkyl alcohol in an amount such that said lower alkyl alcohol constitutes from about 10% to about 80% by volume of said suspension. to

26. A method in accordance with claim 21 in which said solute (ii) is a reducing agent at a concentration of at least about 0.1 g per liter of said aqueous solution.

27. A method in accordance'with claim 26 in which said reducing agent is a member selected from the group consisting of dithiothreitol, glutathione, 2-mercaptoethanol, sodium bisulfite, sodium sulfite, stannous chloride, and manganese chloride.

28. A method in accordance with claim 21 in which said solute (ii) is an oxidizing agent at a concentration of at least about 0.1 g per liter of said aqueous solution.

29. A method in accordance with claim 21 in which said 25 solute (ii) is a high molecular weight polymer at a concentration of at least about 0.1 g per liter of said aqueous solution.

A method in accordance with claim 30 in which said high molecular weight polymer is a polyethyleneglycol.

31. A method in accordance with claim 21 in which said solute (ii) is a hydrogen acceptor at a concentration of at least about 0.1 g per liter of said aqueous solution.

32. A method in accordance with claim 32 in which said hydrogen acceptor is NADP.

33. A method in accordance with claim 21 in which said solute (ii) is a sulfur-containing ring compound at a concentration of at least about 0.1 g per liter of said aqueous solution. 68

34. A method in accordance with claim 34 in which said sulfur-containing ring compound is thioctic acid.

A method for the preparation of an aqueous suspension of monodisperse particles of albumin having a size range of from about 50 to about 5000 nanometers in diameter and stable against resolubilization, said method comprising: treating an aqueous solution of albumin with a lower alkyl alcohol in an amount sufficient to cause said aqueous %o solution to become turbid; and adding to said turbid solution a stabilizing agent selected from the group consisting of oxidizing agents, reducing agents, high molecular weight polymers, hydrogen acceptors, sulfur-containing ring compounds and combinations is thereof in a resolubilization suppressing amount.

36. A method in accordance with claim 36 in which said alcohol is a member selected from the group consisting of methanol, ethanol, n-propanol, isopropanol and n-butanol.

37. A method in accordance with claim 36 in which said alcohol is a member selected from the group consisting of ethanol and n-butanol.

38. A method in accordance with claim 36 in which step (a) comprises adding said lower alkyl alcohol in an amount such that said lower alkyl alcohol constitutes from about 10% to about 80% by volume of said suspension. 9 0

39. A method in accordance with claim 36 in which said stabilizing agent is a reducing agent at a concentration of at least about 0.1 g per liter of said aqueous solution.

A method in accordance with claim 36 in which said reducing agent is a member selected from the group consisting of dithiothreitol, glutathione, 2-mercaptoethanol, sodium- bisulfite, sodium sulfite, stannous chloride, and manganese chloride.

41. A method in accordance with claim 36 in which said stabilizing agent is an oxidizing agent at a concentration of at least about 0.1 g per liter of said aqueous solution. 69

42. A method in accordance with claim 36 in which said stabilising agent is a high molecular weight polymer at a concentration of at least about 0.lg per litre of said aqueous solution.

43. A method in accordance with claim 36 in which said high molecular weight polymer is a polyethyleneglycol.

44. A method in accordance with claim 36 in which said stabilising agent is a hydrogen acceptor at a concentration of at least about 0.1g per litre of said aqueous solution.

A method in accordance with claim 36 in which said hydrogen acceptor is NADP.

46. A method in accordance with claim 36 in which said stabilising agent is a sulfur- containing ring compound at a concentration of at least about 0.1g per litre of said aqueous solution.

47. A method in accordance with claim 36 in which said sulfur-containing ring compound is thioctic acid. 15

48. A particle capable of co-aggregation with platelets, and of aggregation in a solution containing soluble fibrinogen at a concentration of soluble fibrinogen not capable by itself of formation of a clot upon activation by thrombin, said particle comprising a biocompatible matrix and having fibrinogen or RDG-containing peptides within or on the surface of said particle.

49. A particle according to claim 48 where the biocompatible matrix is made of polypeptides.

50. A particle according to claim 48 where the biocompatible matrix is made of lipid containing material.

51. A particle according to claim 48 where the biocompatible matrix is made of o* 25 nucleic acids.

52. A particle according to claim 48 where the biocompatible matrix is made of carbohydrates.

53. A particle according to claim 48 where the biocompatible matrix is made of polysaccharides.

54. A particle according to claim 53 where the biocompatible matrix is made of polysaccharide selected from the group consisting of cellulose, agarose, hemicellulose, starch, mannans, glucans, xanthans, pullutans, arabinans, arabinogalactans, arabinoglucans, arbinoglucoronomannans, xylans, arabinoxylans, carrageenans, colominic acid, and glycosaminoglycans.

55. A particle according to claim 48 where the fibrinogen is covalently bound to the particle.

56. A particle according to claim 48 where the fibrinogen is non-covalently attached to the particle.

57. A particle according to claim 48 where the biocompatible matrix is made of polytactic acid. [n:\Iibc]03046:MEF

58. A method for shortening bleeding time and for decreasing blood loss by intravenous injection, said method comprising administering to a subject a particle comprising a biocompatible matrix with fibrinogen or RDG-containing peptides within or on the surface of said particle, said biocompatible matrix selected from the group consisting of proteins, lipids, nucleic acids, and carbohydrates.

59. The method of claim 58, wherein the biocompatible matrix comprises albumin.

The method of claim 58, wherein the albumin is crosslinked with glutaraldehyde.

61. A method for forming an aggregate inside blood vessels only at the site of a o1 wound due to the action of thrombin, said method comprising administering to a subject a particle comprising a biocompatible matrix with fibrinogen or RDG-containing peptides within or on the surface of said particle, said biocompatible matrix selected from the group consisting of proteins, lipids, nucleic acids, and carbohydrates.

62. The method of claim 61, wherein the biocompatible matrix is a protein, 15 comprising albumin.

63. The method of claim 62, wherein the albumin is crosslinked with I glutaraldehyde.

64. A suspension of particles of albumin, substantially as hereinbefore described with reference to any one of the Examples.

65. A method for the preparation of an aqueous suspension of monodisperse S particles of albumin, substantially as hereinbefore described with reference to any one of the Examples.

66. A particle capable of co-aggregation with platelets, and of aggregation in a solution containing soluble fibrinogen at a concentration of soluble fibrinogen not capable 25 by itself of formation of a clot upon activation by thrombin, substantially as hereinbefore described with reference to any one of the Examples. Dated 20 July, 2000 HemoSphere, Inc. Patent Attorneys for the Applicant/Nominated Person SPRUSON FERGUSON [n:\libc]03046:MEF