Fibrinogen-coated particles for therapeutic 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 with the blood stream. Drugs injected intravascularly reach peak serum levels within a short time, however. Toxic effects can result from such high serum 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 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 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 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 within the intravascular compartment.

Similar advantages by using small particulate carriers for contrast material for CAT scans and nuclear magnetic resonance (NMR) 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 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.

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. Pat. 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. Pat. 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. Pat. 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 Ser. No. 08/212,546, now U.S. Pat. No. 5,616,311.

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

U.S. Pat. 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).

U.S. Pat. No. 3,663,685, Evans et al., May 16, 972, 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 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.

U.S. Pat. No. 5,049,322, Devissaguet, et al., 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 35).

“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 35). Remington does not specially disclose the use of any particular 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 (i.e., 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 noncrosslinked 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 (i.e. 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 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 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 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 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 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 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 (i.e. polyethylene glycol), hydrogen acceptor molecules (i.e. NADP), carboxylic acids having multiple oxygen functionality, and sulfur containing ring compounds (i.e. 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 alpha-keto 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-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 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 water-containing 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 to a biological fluid, or a fluid which is compatible with a biological fluid. Compatible fluids are those which do not cause adverse physiological effects upon administration. 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 volume increase of at least about 50%. The invention is 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 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 50%.

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 albumin-based 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

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 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 1.0

Size (and

mL before addition of alcohol)

Condition) of

HSA

SLS

Particles

Tube

(mL at 80

(mL at 8

Ethanol

Formed

Effect of

No.

mg/mL)

mg/mL)

(mL)

(μm)

Dilution

30

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)

—

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, N.S.) 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 diameter.

Aliquots of the suspension were dispensed into tubes (0.5 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 μL 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 (e.g. 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 (1.5 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 5 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, 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

(μM)

Agent

Minimum

Maximum

Sodium Bisulfite*

650

5000

(NaHSO

3

)

Sodium Sulfide*

500

2500

NADP*

250

2500

DTT*

100

1000

Glutathione* (reduced

100

2500***

form)

PEG (MW 2000)*

12.5

2500

μg/mL

PEG (MW 5000)*

12.5

2500

μg/mL

PEG (MW 8000)*

12.5

2500

μg/mL

Sodium Bisulfite

50

500

Sodium Sulfite-

50

1000

(Na

2

SO

3

)

2-Mercaptoethanol

10**

5000

DL-Lactic Acid

500

1000

Thioctic Acid

10

500

(oxidized)

Stannous Chloride

250

2500

Succinic Acid (4*)

1000

2500

Ascorbic Acid (4*)

1000

2500

1-Ketoglutaric Acid

500

1000

(4*)

Cysteine (4*)

500

2500

Manganese Chloride

1000

2500

(4*)

*Incubation time between agent and sphere suspension was 2 hours, all other incubation times were approximately 18 hours.

**Lowest final concentration tested was 10 μM.

***Highest final concentration tested was 2500 μM.

4*Initial conc of HSA was 5%, 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

(μM, unless

specified)

Agent

lowest

highest

NaNO

2

(sodium

10

10,000

nitrite)

NADH

50

10,000

NADPH

50

10,000

Glutathione (oxidized

50

10,000

form)

Hydrogen peroxide

0.01%

0.3%

Sodium Glucuronate

10

10,000

Isocitric

10

10,000

2-Hydroxybutyric Acid

10

10,000

Glucose

10

10,000

Fructose

10

10,000

Lactose

10

10,000

Galactose

10

10,000

Cytochrome C

10

10

Phenazine

10

10,000

Methosulfate

Dichlorophenolindophe

10

100

nol

FAD

10

500

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 μL 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

FINAL CONCENTRATION OF AGENT

INTERACTION

500 μM

1000 μM

AGENT

TIME (hr)

Time for solution to clarify

Sodium

0.17

<1 min

1 min

Bisulfite

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 10 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 5

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 (15t in normal saline), was added 30 μL of one of the following agents: sodium bisulfite (0.025 to 0.25 M), DTT (0.025 to 0.1 M), NADP (0.05 to 0.1 M), thioctic acid (oxidized, 0.01 to 0.025 M) and control solution (normal saline).

Ethanol (70%) 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 of 30 μL 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 μL of the above mentioned agents were added to 1.0 mL of HSA (15%) 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 hydrogen—accepting (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 (15%) to produce turbidity. To aliquots of 1.0 mL of protein particle suspension was added 5 μL 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.

TABLE V

Stabilization Of Protein Particles Using Combinations of

Hydrogen-Accepting and Hydrogen-Donating Agents

After addition of

Tube

NADP

NADPH

N.S.

21

0

0

clear

22

0

0.1

clear

23

0.1

0

turbid

24

0.1

0.10

turbid

25

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

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

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

Ethanol (70%) was added to the HSA solutions until the solutions became turbid. Thereafter, 10 μL 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, 5μL 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 (15%). Ethanol (70%) was subsequently added to produce protein spheres. Within 5 minutes, 5 μL 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 did not result in substantial loss of enzyme activity.

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.

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 37° C. 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 10

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 (5 μL 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 (15%) to provide a concentration of 5 μg of DNA per mL. Control spheres were formed with HSA without DNA. Ethanol (70%) was added to produce turbidity. The spheres thus formed were stabilized by incubating the mixtures overnight with 5 μL of sodium bisulfite (0.1 M) per mL suspension.

The spheres were then washed in normal saline (3×10 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 prepared from DNA-containing HSA solutions, but not from control spheres or the supernatants of both experimental and 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 spheres during the stabilizing phase.

The experimental protocol of Example 12 was followed except that protein particles were first formed by addition of ethanol (70%) to 1.0 mL of HSA (15%) in the absence of DNA or RNA (with RNAase inhibitor). Within 0.5 min of addition of the stabilizing agent (sodium bisulfite 0.1 M), 5 μL of a suspension of DNA or RNA was added to the protein spheres to result in a final concentration of 5 μg 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. 20 microliters of a 1M or 1 mM solution of the potential stabilizing agent was added to 2 mL of sphere suspensions. Only two final concentrations (10 mM 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:

ammonium chloride

ammonium nitrate

ammonium sulfate

ammonium phosphate, monobasic and dibasic

potassium iodide

potassium acetate

potassium bicarbonate

molybdic acid, sodium salt

Cations:

cobalt chloride

cupric chloride

magnesium chloride

manganese chloride

ferric nitrate

Vitamins:

cyanobalamin (B

12

)

ergocalcifero (D

2

)

Acids:

folinic acid

δ-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

chloramphenicol

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:

Minimal Final Concentration

Effective Chemicals

(mM)

magnesium chloride

5

mM

manganese chloride

0.5

mM

γ-aminolevulinic acid

0.002

mM

gentamycin

0.05

mg/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 sulfoxide

1/100

dilution

Samples of suspensions were examined under the microscope and. found to consist of monodispersed spheres with no aggregates seen.

EXAMPLE 15

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 pre-incubated 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 1mL of 15% HSA with 25 μL 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 3× volume (7.5 mL) 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

Adding

normal saline 1 minute

normal saline 60 minutes after

Incubation

after forming spheres

forming spheres

Time

0.05 M

0.1 M

0.05 M

0.1 M

(minutes)

NaHSO

3

NaHSO

3

NaHSO

3

NaHSO

3

5

immediately

immediately

2 hours

more than 8 hours

10

immediately

immediately

2.5 hours

more than 8 hours

20

immediately

immediately

2.5 hours

more than 8 hours

30

immediately

immediately

2.5 hours

more than 8 hours

60

immediately

immediately

2.5 hours

more than 8 hours

120

immediately

immediately

2 hours

more than 8 hours

Control

immediately

immediately

(no

NaHSO

4

)

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

I. 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.

5. Add 3× 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:

After

NaHSO

4

Concentration

Adding

(μM)

Appearance After 2 Hours

Normal

In Ethanol

In Suspension

Gross

Microscopic

Saline

50,000

30,000

gelatinous

aggregates

precipitation

25,000

15,000

gelatinous

aggregates

precipitation

12,500

7,500

gelatinous

aggregates

precipitation

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, i.e., 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 (e.g., 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 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

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 adding 70% ethanol to 15% HSA,

2. The final Na bisulfite concentration in all three methods .will be about lmM.

3. Add 0.5× 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 (× 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.)

30

(Part of the spheres dissolved after adding

fibrinogen. All spheres then dissolved during

the washing process.)

60

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

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-6f 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.

5. 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 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 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 co-aggregation with platelets or in improving bleeding time. These are as follows:

(1) 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.

(2) 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 the wound, but more importantly to prevent the formation 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.

(c) 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 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.

(d) It was also found that upon activation, platelets release ATP and serotonin. In a “thrombocytopenic” (i.e., 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 not 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, because they are more concentrated where needed. Fibrinogen-coated 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 non-covalently to the surface of the particles.

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 (e.g., 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. Pat. Nos. 4,904,479 and 4,728,578), polyesters and/or polyamides encapsulated in methylcellulose (U.S. Pat. No. 5,233,995), gelatin, albumin, collagen (U.S. Pat. No. 4,844,882), aliphatic and alicyclic carbon-containing compounds (U.S. Pat. No. 5,143,716), polyaminoacids (U.S. Pat. No. 4,247,406), e.g., polyglutamate, and polylysine either separately or in combination with proteinaceous microspheres (e.g., albumin EP 0 633 030, WO 92/17213, WO 91/12823 synthetic polymers (WO 92/17514, U.S. Pat. Nos. 4,089,800, 4,572,869, 3,429,827, polyesters, e.g., polymers and copolymers polylactide/polyglycolide and polylactones (e.g., ∈-caprolactone (U.S. Pat. No. 4,637,605, Wo 92/18164) polysilicones (G.B. 2,026,513B), cyclodextrins (GB 2,090,738B, WO 93/02712, WO 92/21382) non-proteinaceous cross-linked or polymerized amphophilic moieties (WO 92/17212), hydrophilic synthetic polymers such as polyalkylene glycols (e.g., PEG), polyvinylpyrrolidones (U.S. Pat. No. 5,470,911).

EXAMPLE 18

Thrombospheres (TS) are cross-linked human serum 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 (BT), Blood Loss (BL) and on platelet survival following infusion of TS. Similar results are obtained with other fibrinogen coated particles (crosslinked or non-crosslinked), particularly albumin particles, where the fibrinogen is adsorbed non-covalently onto the surface.

Methods

Severely thrombocytopenic rabbits (av plt count <10×10

3

/ul) received i.v., either TS, 7.5×10

9

/kg; control albumin spheres (CS) which had no fibrinogen, 7.5×10

9

/kg; or an equal volume of normal saline (NS). 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 (1 h)

BT (24 h)

BT (48 h)

BT (72 h)

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 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 (i.e., without 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 (i.e., those with some digested fibrinogen (fibrin) on their surface), 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 one or both of the following mechanisms:

(1) 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 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.

(2) 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 time 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:

(i) 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.

The conditions and diseases treated in the present invention include thrombocytopenia, reducing bleeding time and blood loss, ameliorating platelet dysfunction due to kidney failure, drug sensitivity, drug action (e.g., aspirin, or antiplatelet antibodies) or as a result of cardiopulmonary 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, i.e., 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, e.g., from about 0.01 percent weight (%w) to about 99.99%w of the drug based on the total formulation and about 0.01%w to 99.99%w excipient. Preferably the drug is present at a level of about 10%w to about 70%w.

Generally, an acceptable daily dose is of about 0.001 to 50 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 (i.e., 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 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

15. alpha 2-macroglobulin

16. streptokinase

17. anti-progesterone antibody

18. anti-leukotriene B4 antibody

19. CGGRGDF-NH

2

20. doxorubicin

21. daunarubicin

22. EDTA-conjugated to HSA

23. DTPA-conjugated to HSA

24. technetium

25. 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

30. HSA conjugated to biotin

31. alkaline phosphatase

32. peroxidase

33. amphotericin B

34. Adjuvant peptide (N-acetylmuramyl-l-alanyl-d-isoglutamine)

35. HIV-1 protease substrate (acetyl-ser-gln-asn-tyr-pro-val-val-amide)

36. Fe

3

O

4

magnetite or magnetic particles

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 (TcO

4

−

) 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 TcO

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 TcO

2

−

before it binds to 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, i.e., one which is not yet associated with the particles, then binding the Tc99m-chelating agent 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, 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-10-pentadecenoic, 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-11-octadecenoic, trans-11-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-octadecadienoic, cis-9,12,15-octadecadienoic, cis-6,9,12,15-octadecatetraenoic, cis-10-nonadecenoic, cis-5-eicosenoic, cis-8-eicosenoic, cis-1l-eicosenoic, cis-13-eicosenoic, cis-11,14eicosadienoic, cis-5,8,11-eicosatrienoic, 5,8,11-eicosatriynoic, cis-8,11,14eicosatrienoic, cis-11,14,17-eicosatrienoic, cis-5,8,11,14-eicosatetraenoic, 5,8,11,14eicosatetraynoic, cis-5, 8, 11 14, 17-eicosapentaenoic, cis-13-docosenoic, trans-13docosenoic, cis-13,16-docosadienoic, cis-13,16,19-docosatrienoic, cis-7,10,13,16-docosatetraenoic, cis-4,7,10,13,16,19-docosahexaenoic, cis-15-tetracosenoic acids.

Lectins

Abrus precatorius

(Agglutinin, Abrin A toxin, Abrin C toxin),

Agaricus bisporus, Anguilla anguilla, Arachis hypogaea, Bandeiraea simplicifolla

(BS-I, BS-I-B4, BS-I-AB3, BS-I-A2B2, BS-I-A3B, BS-I-A4, BS-II),

Bauhinia purpurea, Caragana arborescens, Cicer arietinum, Codium fragile,

Concanavalin A, SuccinylConcanavalin A,

Datura stramonium, Dolichos biflorus, Erythrina corallodendron, Erythrina cristagalli, Euonymus europaeus,

Glycine max,

Helix aspersa, Helix pomatia, Lathyrus odoratus, Lens culinaris, Limulus polyphemus

(Bacterial agglutinin),

Lycopersicon esculentum, Maclura pomifera, Momordica charantia, Mycoplasma gaillsepticum, Naja mocambique mocambique, Naja naja kaouthia, Perseau americana, 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, RCA60, Toxin, RCA120),

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 Combinations of Several Proteins

Clq, C2, C3, C4, C5, C6, C7, C8, C9, Properdin factor B.

Spin Labels and Spin Traps

Doxyl Nitroxides, e.g., 3-beta-doxyl-5-alpha-cholestane;

Proxyl Nitroxides, e.g., 3-(4-nitrophenoxycarbonyl)-proxyl;

Tempo Nitroxides, e.g., Tempo;

DL-t-Butyl Nitroxide;

Spin traps: Nitrosobenzene, Nitrosadisulfonic acid, 2-methyl-2-Nitroso-Propane.

Arachidonic Acid Cascade and Related Compounds

HETEs: 5(S)-HETE[5(S)-hydroxy-6-trans-8-cis-11-cis-14-cis-eicosatetraenoic acid]; 11 (S)-HETE[11 (S)-hydroxy-5-cis-8-cis-12-trans-14-cis-eicosatetraenoic acid]; 12(R)-HETE[12(R)-hydroxy-5-cis-8-cis-10-trans-14-cis-eicosatetraenoic acid]; 12(S)-HETE[12(S)-hydroxy-5-cis-8-cis-lo-trans-14-cis-eicosatetraenoic acid]; 15(S)-HETE[15(S)-hydroxy-5-cis-8-cis-11-cis-13-trans-eicosatetraenoic acid];

HPETEs: 5(S)-HPETE [5(S)-hydroperoxy-6-trans-8-cis-11-cis-14-cis-eicosatetraenoic acid]; 12(S)-HPETE[12(S)-hydroperoxy-5-cis-B-cis-10-trans-14-cis-eicosatetraenoic acid]; 15(S)-HPETE [15(S)-hydroperoxy-5-cis-8-cis-11-cis-13-trans-eicosatetraenoic acid];

DiHETEs: 5(S),6(R)-DiHETE[5(S),6(R)-dihydroxy-7-trans-9-trans-11-cis-14-cis-eicosatetraenoic acid]; 5(S),12(S)-DiHETE[5(S),12(S)-dihydroxy-6-trans-8-cis-10trans-14-cis-eicosatetraenoic acid]; 5(S) ,15(S)-DiHETE[5(S),15(S)-dihydroxy-6-trans-8-cis-11-cis-13-trans-eicosatetraenoic acid]

Other Arachidonic Acid Cascade Related Compounds

13-Azaprostanoic acid; Baicalein; 7-7-dimethyleicosadienoic acid; 5,8,1leicosatriynoic 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, F2α, I2, G2, H2); 16-16-Dimethyl-prostaglandin E2; 6-Keto-prostaglandin F1α; 2,3-Dinor 6-keto-prostaglandin F1α; 9,11-Dideoxy-9α, 11α-methanoepoxyprostaglandin-F2α; carbacyclin; 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, Brevetoxin (PbTx-1, 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 fragments

N-Acetyl-Hirudin and its fragments

Acetyl-Leu-Leu-Argininal

N-Acetyl-Leu-Leu-Methioninal

N-Acetyl-Leu-Leu-Norleucinal

Acetyl-Met-Asp-Arg-Val-Leu-Ser-Arg-Tyr

N-Acetyl-Met-Leu-Phe

N-Acetylmuramyl-D-alanyl-D-isoglutamine

N-Acetylmuramyl-L-alanyl-D-isoglutamine

N-Acetylmuramyl-L-alanyl-L-isoglutamine

N-Acetylmuramyl-Ala-D-isoglutaminyl-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-His-Phe-Arg-Trp-Gly-Lys-Pro-Val Amide

N-Acetyl-Thr-Ile-Nle-Phe(CH

2

NH)-Nle-Gln-Arg Amide

ACTH (Adrenocorticotropic Hormone)

Adrenal Cyclase Activating Polypeptide-27

Adrenal Medulla Peptides

Adrenal Peptide E

Adrenocorticotropic Hormone and fragments

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-gamma-Glu-Lys-D-Ala-D-Ala

Ala-Gly-Cys-Lys-Asn-Phe-Phe-Trp-Lvs-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

Ala-Ser-His-Leu-Gly-Leu-Ala-Arg

beta-Ala-Ser-His-Leu-Gly-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

Alytesin

Amastatin

beta-Amyloid 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

Anthranilyl-His-Lys-Ala-Arg-Val-Leu-p-Nitro-Phe-Glu-Ala-Nle-Ser Amide

Antide

Anti-inflammatory Peptide 1

Antipain

Antireproductive Tripeptide

Apamin

Arg-Arg-Leu-Ile-Glu-Asp-Ala-Glu-Tyr-Ala-Ala-Arg-Gly

Arg-Arg-Leu-Ile-Glu-Asp-Asn-Glu-Tyr-Thr-Ala-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-Gly-Phe-Phe-Tyr-Thr-Pro-Lys-Ala

Arg-Gly-Pro-Phe-Pro-Ile

Arg-His-Phe

Arg-Lys-Arg-Ala-Arg-Lys-Glu

Arg-Lys-Asp-Val-Tyr

Arg-Lys-Glu-Val-Tyr

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-Val-Val-Leu-Pro-Arg-Pro-Val-Cys-Phe-Glu-Lys-Gly-Met-Asn-Tyr-Thr-Val-Arg

Asn-Ala-Gly-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-Ile-Asp-Ser-Phe-Gln-Glu-Ile-Val

Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-His-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-(Benzyl)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-BOC-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

Bradykinin and analogs

Bradykinin Potentiator (e.g. 5a, 9a, B, C)

Brain Natriuretic Peptide

Brefeldin A

Buccalin

Bursin

Caerulein

Calcitronin

Calcitonin Gene Related Peptide

beta-Calcitonin 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

Cardioexcitatory Peptide

alpha-Casein and fragments

Beta-Casomorphin

Na-CBZ-Arg-Arg-Pro-Phe-His-Sta-Ile-His-Ne-BOC-Lys Methyl

Ester

CBZ-Leu-Val-Gly Diazomethyl Ketone

N-CBZ-D-Phe-Phe-Gly

N-CBZ-Pro-D-Leu

N-CBZ-Pro-Leu-Gly Hydroxamate

CD4 and fragments

Cecropins

Cerebellin

Chemostactic Peptides

Cholecystokinin and fragments

Chorionic Gonadotropin and fragments

Chromostatin-20

Chymostatin

Circumsporozoite (CS) Protein of Plasmodium falciparum

repetitive sequences

Collagen

Conotoxin GI

μ-conotoxin GIIIB

ω-conotoxin GVIA

α-conotoxin SI

Copper Binding Peptide

Corazonin

Corticotropin A

Corticotropin-Like Intermediate Lobe Peptide

Corticotropin Releasing Factor and analogs

Tyr-Corticotropin Releasing Factor

Corticotropin Releasing Factor Antagonist

C-Peptide and fragments

Cyclic-AMP Dependent Protein Kinase Substrate

Cyclo(7-Aminoheptanoyl-Phe-D-Trp-Lys-Thr[Bzl])

Cyclo(D-Asp-Pro-D-Val-Leu-D-Trp)

Cyclohexylacetyl-Phe-Arg-Ser-Val-Gln Amide

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-Ile-Lys-Val-Ala-Val-Ser-Ala-Asp-Arg

Cys-Tyr-Ile-Gln-Asn-Cys-Pro-Arg-Gly Amide

Cys-Tyr-Ile-Gin-Asn-CYs-Pro-Leu Gly Amide

Cys-TYr-Ile-Ser-Asn-Cys-Pro-Ile-Gly Amide

DAGO (D-Ala2, N-Me-Phe4-Gly5-ol-Enkephalin)

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.

Endothelin

alpha-Endorphin

beta-Endorphin and fragments

gamma-Endorphin

Endothelins

Enkephalin, Leucine and analogs

Enkephalinamide, Leucine and analogs

Enkephalin, Methionine and analogs

Enkephalinamide, Methionine and analogs

Enzyme Inhibitors

Eosinophilotactic Tetrapeptides

Epiamastatin

Epibestatin

Epidermal Growth Factor

Epidermal Mitosis Inhibiting Pentapeptide

Experimental Allergic Encephalogenic Peptide

Erythropoietin fragment 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 (e.g. 50K)

Fibronectin Chymotrypsin (e.g. 40K, 45K, 120K)

Fibronectin Trypsin (e.g. 30K, 60K)

N-FMOC-val-Gly-Gly-O-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

Gastrin-Tetrapeptide Amide

Gastrointestinal Peptides

Gilodeliquescin

Gin-Ala-Thr-Val-Gly-Asp-Ile-Asn-Thr-Glu-Arg-Pro-Gly-Met-Leu-Asp-Phe-Thr-Gly-Lys

Gln-AJa-Thr-val-Gly-Asp-Val-Asn-Thr-Asp-Arg-Pro-Gly-Leu-Leu-Asp-Leu-Lys

Gln-Arg-Arg-Gln-Arg-Lys-Ser-Arg-Arg-Thr-lle

Gln-Lys-Arg-Pro-Ser-Gln-Arg-Ser-Lys-Tyr-Leu

Glu-Ala-Glu

Glu-Ala-Glu-Asn

Glucagon

Glucagon(1-37)

Glucagon-Like Peptide I and fragments

Glu-Leu-Ala-Gly-Ala-Pro-Pro-Glu-Pro-Ala

Glutathione and analogs

Gly-Arg-Ala-Asp-Ser-Pro

Gly-Arg-Ada-Asp-Ser-Pro-Lys

Gly-Arg-Gly-Asp

Gly-Arg-Gly-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-Gln

Gly-Glu-Gln-Arg-Lys-Asp-Val-Tyr-Val-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-Val-Gly

Gly-Pro-Arg

Gly-Pro-Arg-Pro

Gly-Pro-Gly-Gly

Granuliberin R

Granulocyte Nacrophage-colony Stimulating Factor

GRF 1-40, Human

Growth Hormone Release Inhibiting Factor

Growth Hormone Releasing Factor and fragments

H-142

Helodermin

Hepatitis A viral proteins and peptides

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-Val-Leu-Asp-Leu

His-Leu-Gly-Leu-Ala-Arg

His-Lys-Ala-Arg-Val-Leu-p-Nitro-Phe-Glu-Ala-Nle-Ser Amide

D-His-Pro-Phe-His-Leu-ph-[CH

2

NH]-Leu-Val-Tyr

Histones

HIV Envelope Protein (gp4l) fragment 579-601

HIV Envelope Protein (gp120) fragments

HIV Protease Inhibitor

HIV Substrate, III

HIV viral protein and peptides

Histone H2A fragment 86-120

Hydra Peptide and fragments

Hypercalcemia Malignancy Factor -40

Hypertrehalosaemic Neuropeptide

Iberiotoxin

Ile-Pro-Ile

Ile-Val-Pro-Phe-Leu-Gly-Pro-Leu-Leu-Gly 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 (e.g. alpha 4, alpha V beta 5 alpha2, alpha3, alpha 5, alpha V, beta 1, beta 2, beta 4)

Interleukin 1B fragment (163-171)

Interleukin-2 Receptor C-Terminal Sequence

Interleukin (e.g. 1 alpha, 2, 6, gamma)

Isotocin

Kallidin

Kallikrein Inhibitor

Kassinin

Katacalcin and analogs

Kemptide and analogs

Kentsin

Kinetensin

Kyotorphin and analogs

Laminin and fragments (929-933)

Leu-Arg-Arg-Ala-Ser-Leu-Gly

Leu-Arg-Arg-Ala-Hse-Leu-Gly

Leu-Arg-Arg-Trp-Ser-Leu-Gly

Leucokinins

Leucopyrokinin and fragments

Leu-Leu Methyl Ester

Leu-Lys-Lys-Phe-Asn-Ala-Arg-Arg-Lys-Leu-Lys-Gly-Ala-Ile-Leu-Thr-Met-Leu-Ala

Leu-Pro-Pro-Ser-Arg

Leu-Ser-(pNO2)-Phe-Nle-Ala-Leu Methyl Ester

Leupeptin

LH-RH (Luteinizing Hormone Releasing Hormone) and analogs

beta-Lipotropin and fragments

Litorin

Lys-Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg

Lys-Ala-Arg-Val-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-Pro-Pro-Glu-Pro-Glu-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 HR1

Mast Cell Degranulating Peptide HR2

Mastoparan

Alphal-Mating Factor

MCD Peptide

MB-35

Alpha-Melanocyte Stimulating Hormone and analogs

Beta-Melanocyte Stimulating Hormone

Delta-Melanocyte Stimulating Hormone

Melittin

Merosin

Met-Asn-Tyr-Leu-Ala-Phe-Pro-Arg-Met Amide

Met-Gln-Met-Lys-Lys-Val-Leu-Asp-Ser

Met-Gly-Trp-Asn-Ser-Thr-Thr-Phe-His-Gin-Thr-Leu-Gln-Asp-Pro-Arg-Val-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-Cys-Tyr-D-TrP-Lys-Val-Cs-Thr Amide

coendorphin

Beta-Neoendorphin

Alpha-Neurokinin

(Ala5, Beta-Ala8)-alpha-Neurokinin fragment 4-10

Neurokinin (e.g., A, Nle-10, B, MePhe7-B)

Neuromedins (e.g., B,C)

Neuropeptide K

Neuropeptide Y

Neurotensin and analogs

N-Nicotinoyl-Tyr-(Nalpha-CBZ-Arg)-Lys-His-Pro-Ile

Nle-Arg-Phe Amide

Nle-Sta-Ala-Sta

NeutrAvidin

octadecaneuropeptide (e.g. 6, 7, 8)

Osteocalcin fragment 7-19

Osteocalcin fragment 45-49

Oxyntomodulin

Oxytocin and analogs

PACAP27 Amide

Pancreastatin and fragments

Pancreatic Polypeptide

Parathyroid Hormone and fragments

Pardaxin

Pentagastrin

Pepstatin A

Peptide II of T, wagleri Venom

Peptide T

Peptide YY

pGlu-Ala-Glu

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-Asn-Gln-Trp-AJa-Val-Gly-His-Leu-Met

Amide

pGlu-Gln-Asp-Tyr(SO3H)-Thr-Gly-Trp-Met-Asp-Phe Amide

pGlu-Glu-Asp-Ser-Gly

pGlu-Gly-Leu-Pro-Pro-Arg-Pro-Lys-Ile-Pro-Pro

pGlu-Gly-Leu-Pro-Pro-Gly-Pro-Pro-Ile-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-lle-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-Ph6-Gly

Phe-Leu-Arg-Phe Amide

Phe-Leu-Glu-Glu-Ile

Phe-Leu-Glu-Glu-Leu

Phe-Leu-Glu-Glu-Val

Phe-Leu-Phe-Gln-Pro-Gln-Arg-Phe Amide

Phe-Met-Arg-Phe Amide

Phe-Met-Arg-D-Phe Amide

Phe-Met-D-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

Physalaemin

Platelet Derived Growth Factor (AB-chain, heterodimer, AA homodimer, BB homodimer)

Platelet Membrane Glycoprotein IIB Peptide

Pre-Pro-Gonadotropin Releasing Hormone fragment 14-26

Pressinoic Acid

N-proCalcitonin 1-57

Proctolin

Prodynorphin 228-240

Proenkephalin

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-Ala-Ala-Lys-Lys

Prosomatostatin 1-32

PYY

Protected Marine Adhesive Peptide

Protein A

Protein G (binds to Fc region, specially of IgGI subclass)

Protein Kinase C

Protein Kinase C Substrate

Protein Kinase Inhibitor

Pro-Thr-Pro-Ser Amide

PTH

PTH-Related Protein (1-40)

Ranatensin

Renin Inhibitors

Renin Substrate Tetradecapeptide

N-(alpha-Rhamnopyranosyloxyhydroxyphosphinyl)-Leu-Trp

Sarafotoxin

Schizophrenia Related Peptide

secretin

Senktide

Ser-Asp-Gly-Arg-Gly

Ser-Gln-Asn-Phe-Phi(CH

2

N)-Pro-Ile-Val-Gln

Ser-Gin-Asn-Tyr-Pro-Ile-Val

Ser-Ile-Gly-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

Speract

Streptavidin

Streptolysin

Substance P and analogs

SV40 Tumor Antigen C-Terminal Sequence

Syndyphalin

Syntide

Tenascin

Terlipressin

Thapsigargin

DL-Thiorphan

Thr-Lys-Pro-Arg

Thrombin Receptor Activator

Thr-Phe-Gln-Ala-Tyr-Pro-Leu-Arg-Glu-Ala

Thr-Pro-Arg-Lys

Thr-Ser-Lys

Thr-Thr-Tyr-Ala-Asp-Phe-Ile-Ala-Ser-Gly-Arg-Thr-Gly-Arg-Arg-Asn-Ala-Ile-His-Asp

Thr-Tyr-Ser

Thr-Val-Leu

Thrombospondin

Thymopoietin fragments

Thymosin

Thymosin fragments

Thyrocalcitonin

Thyrotropin Releasing Hormone and Related Peptides

Tocinoic Acid

Toxin, Snake

TP-5

Transforming Growth Factor-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-Met-Asp-Phe Amide

Trp-Nle-Arg-Phe Amide

Tuftsin and analogs

Tumor Necrosis factor (e.g. alpha) Tyr-Ala-Gly-Ala-Val-Val-Asn-Asp-Leu

Tyr-D-Ala-Phe-Asp-Val-Val-Gly Amide

Tyr-D-Ala-Phe-Glu-Val-Val-Gly Amide

Tyr-Gly-Ala-Val-Val-Asn-Asp-Leu

Tyrosine Protein Kinase Substrate

Tyr-D-Ala-Gly

Tyr-Gly-Gly

Tyr-D-Ala-Phe-Met Amide

Tyr-Arg

Tyr-Gly-Gly

Tyr-Gly-Gly-Phe-Leu

Tyr-Gly-Gly-Phe-Leu-Arg-Arg-Ile-Arg-Pro-Arg-Leu-Arg-Gly-Aminopentylamide

Tyr-Gly-Gly-Phe-Met-Arg-Arg-Val

Tyr-Gly-Gly-Phe-Met-Arg-Arg-Val Amide

Tyr-Gly-Gly-Phe-Met-Arg-Gly-Leu

Tyr-Gly-D-Trp-Phe-D-Phe Amide

Tyr-Ile-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-Gly-Phe-Leu-Thr

Tyr-D-Trp-Ala-Trp-D-Phe Amide

Tyr-D-Trp-Ala-Trp-D-Phe Methyl Amide

Urodilatin

Urotensin I

Urotensin II

Val-Ala-Ala-Phe

Val-Arg-Lys-Arg-Thr-Leu-Arg-Arg-Leu

Val-Glu-Glu-Ala-Glu

Val-Glu-Pro-Ile-Pro-Tyr

Val-Glu-Ser-Ser-Lys

Val-Gly-Asp-Glu

Val-Gly-Ser-Glu

Val-Gly-Val-Ala-Pro-Gly

Val-His-Leu-Thr-Pro

Val-His-Leu-Thr-Pro-Val-Glu-Lys

Val-Ile-His-Asn

Valosin

Val-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-Acetamido-2-deoxy-3-O-Beta-D-galactopyranosyl-beta-D-glucopyranoside-HSA Conjugate

Carboxyethylthioethyl 2-Acetamido-2-deoxy-4-O-beta-D-galactopyranosyl-beta-D-glucopyranoside-HSA Conjugate

Carboxyethylthioethyl 2-Acetamido-4-O-(2-acetamido-2-deoxy-beta-D-glucopyranosyl-6-O-(alpha-L-fucopyranosyl))-2-deoxy-beta-D-glucopyranoside-HSA Conjugate

Carboxyethylthioethyl 4-O-alpha-D-Galactopyranosyl-beta-D-galactopyranoside-HSA Conjugate

Carboxyethylthioethyl 4-O-(4-O-(6-O-alpha-D-Glucopyranosyl-alpha-D-glucopyranosyl)-alpha-D-glucopyranosyl)-beta-D-glucopyranoside-HSA Conjugate

Free Oligosaccharides and Simple Derivatives

2-Acetamido-6-O-(2-acetamido-2-deoxy-beta-D-glucopyranosyl)-2-deoxy-D-glucopyranose

2-Acetamido-2-deoxy-3-O-beta-D-galactopyranosyl-D-galactopyranose

2

-Acetamido-2-deoxy-4-O-(4-O-Beta-D-galatopyranosyl-beta-D-galactopyranosyl)-D-glucopyranose

2-Acetamido-2-deoxy-3-O-beta-D-galactopyranosyl-D-glucopyranose

2-Acetamido-2-deoxy-6-O-beta-D-galactopyranosyl-D-glucopyranose

6-O-(2-Acetamido-2-deoxy-4-O-(beta-D-galactopyranosyl)-beta-D-glucopyranosyl)-D-galactopyranose

6-O-(2-Acetamido-2-deoxy-beta-D-glucopyranosyl)-D-galactopyranose

4-O-(6-O-(2-Acetamido-2-deoxy-beta-D-glucopyranosyl)-beta-D-galactopyranosyl)-D-glucopyranose

N-Acetyllactosamine

Benzyl 2-Acetamido-6-O-(2-acetamido-2-deoxy-beta-D-glucopyranosyl)-2-deoxy-alpha-D-glucopyranoside

Benzyl 2-Acetamido-2-deoxy-3-O-beta-D-galactopyranosyl-alpha-D-galactopyranoside

Benzyl 2-Acetamido-2-deoxy-3-O-beta-D-galactopyranosyl-beta-D-glucopyranoside

Benzyl 4-O-beta-D-Galactopyranosyl-beta-D-glucopyranoside

n-Butyl 4-O-beta-D-Galactopyranosyl-beta-D-glucopyranoside

D-(+)-Cellobiose

D-(+)-Cellopentaose

D-(+)-Cellotetraose

D-(+)-Cellotriose

Digalacturonic acid

Ethyl 2-Acetamido-2-deoxy-4-O-(4-O-alpha-D-galactopyranosyl-beta-D-galactopyranosyl)-beta-D-glucopyranoside

Ethyl 4-O-beta-D-Galactopyranosyl-beta-D-glucopyranoside

2′-Fucosyllactose

3-Fucosyllactose

4-O-alpha-D-Galactopyranosyl-D-galactopyranose

6-O-beta-D-Galactopyranosyl-D-galactopyranose

4-O-(4-O-beta-D-Galactopyranosyl-beta-D-galactopyranosyl)-D-Glucopyranose

4-O-beta-D-Galactopyranosyl-D-mannopyranose

4-O-(4-O-(6-O-alpha-D-Glucopyranosyl-alpha-D-glucopyranosyl)alpha-D-glucopyranosyl)-D-glucopyranose

Lacto-N-tetraose

3-O-alpha-D-Mannopyranosyl-D-mannopyranose

Methyl 4-O-(3-O-(2-Acetamido-2-deoxy-4-O-beta-D-galactopyranosyl-beta-D-glucopyranosyl)-beta-D-galactopyranosyl)-beta-D-glucopyranoside

Methyl 3-O-(2-Acetamido-2-deoxy-beta-D-glucopyranosyl)-beta-D-galactopyranoside

Methyl 6-O-(2-Acetamido-2-deoxy-beta-D-glucopyranosyl)-alpha-D-mannopyranoside

Methyl 3,6-Di-O-(alpha-D-mannopyranosyl)-alpha-D-mannopyranoside

Methyl 3-0-beta-D-Galactopyranosyl-beta-D-galactopyranoside

4-O-(2-O-Methyl-beta-D-galactopyranosy)-D-glucopyranose

Methyl 4-O-beta-D-Galactopyranosyl-beta-D-glucopyranoside

Methyl 2-O-alpha-D-Mannopyranosyl-alpha-D-mannopyranoside

Methyl 3-O-alpha-D-Mannopyranosyl-alpha-D-inannopyranoside

Methyl 4-O-alpha-D-Mannopyranosyl-alpha-D-mannopyranoside

Methyl 6-O-alpha-D-Mannopyranosyl-alpha-D-mannopyranoside

n-Propyl 4-O-beta-D-Galactopyranosyl-beta-D-glucopyranoside

Trigalacturonic acid

Activated Oligosaccharides

Carbomethoxyethylthioethyl 2-Acetamido-2-deoxy-4-O-beta-D-galactopyranosyl-beta-D-glucopyranoside

Carbomethoxyethylthioethyl 4-O-(4-O-(6-O-alpha-D-Glucopyranosyl-alpha-D-glucopyranosyl)-alpha-D-glucopyranosyl)-beta-D-glucopyranoside

p-Nitrophenyl 2-Acetamido-2-deoxy-3-O-(2-acetamido-2-deoxy-beta-D-glucopyranosyl)-alpha-D-galactopyranoside

p-Nitrophenyl 2-Acetamido-2-deoxy-3-O-(Beta-D-galactopyranosyl)-alpha-D-galactopyranoside

p-Nitrophenyl 2-Acetamido-2-deoxy-3-O-beta-D-galactopyranosyl-beta-D-glucopyranoside

p-Nitrophenyl 6-O-beta-D-galactopyranosyl-beta-D-galactopyranoside

p-Nitrophenyl alpha-D-Maltopentaoside

Neo-glycolipids

Octadecylthioethyl 4-O-alpha-D-Galactopyranosyl-beta-D-galactopyranoside

Octadecylthioethyl 4-O-(4-O-(6-O-alpha-D-Glucopyranosyl-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; α2-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 (polyI-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-(1-3)-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; primaquin; Dapasone; Spiramycin; piritrexim

Adjuvant Peptides

N-acetylmuramyl-L-alanyl-D-isoglutamine

N-acetylmuramyl-D-alanyl-D-isoglutamine

N-acetylmuramyl-L-alanyl-L-isoglutamine

N-acetylmuramyl-L-alanyl-D-isoglutamine-6-O-stearoyl

N-acetyl-glucosaminyl-beta(1-4)-N-acetylmuranyl-L-alanyl-D-isoglutamine

Components of Freund's Complete Adjuvant

Components of Freund's Incomplete Adjuvant

Dimethyldioctyldecyl Ammonium 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

Lipoxin A4, B4

Lipoxygenase

Prostaglandan Synthetase

Chelating Agents

Iminodiacetic Acid (e.g., dimethyl-ida, paraisopropyl-ida, parabutyl-ida, diisopropyl-ida, diethyl-ida)

EDTA (Ethylenediaminetetraacetic acid)

NTA (Nitriloacetic acid)

TPP (Tripolyphosphate

Cysteine

DEDTC (Diethyldithiocarbamate)

Citric acid

Tartaric acid

Penicillamine

EGTA

Caged Calcium Chelators

NITR5, NITR7, DM-nitrophen, NITRS/AM; Ammonium N-nitrosophenyl-hydroxylamine; Ammonium purpurate; alpha-Benzoin oxime; N,N-Bis-(hydroxyethyl)-glycine; 2,3-butane-dione dioxime; Trans-1,2-Diaminocyclo-hexanetetra-acetic acid (CDTA); Diethylene-triaminopenta-acetic acid (DTPA); 4,5-Dihydroxy-benzene-1,3-disulphonic acid; 2,3-Dimercapto-1-Propanol; Diphenylthio-carbazone; 2,2′-Dipyridyl; 3,6-Disulpho-1,8-dihydroxy-naphthalene; Dithiooxamide; Eriochrome Black T; Ethylene-diamine; Ethylenediaminetetraacetic acid (EDTA); (Ethylene-dioxy)-diethylenedinitrilo-tetraacetic acid (EGTA); o-Hydroxybenzaldehyde oxime; N-(2′-Hydroxyethyl)iminodiacetic acid (HIMDA); 8-Hydroxy-quinoline; 8-Hydroxyquinoline-5-sulphonic acid; 4-Methyl-1,2-dimercapto-benzene; Nitrilotriacetic acid (NTA); 5-Nitro-1,10-phenanthroline; 1,10-Phenanthroline; Potassium ethyl xanthate; Salicylic acid; sodium diethyldithio-carbamate; 2-Thenoyl-2-furoylmethane; Thenoyl-trifluoro-acetone; Thiourea; Triethylenetetramine

Deferoxamine mesylate

Edetate Calcium disodium

meso 2,3-dimercapto succinic acid

Penicillamine

Trientine

Chelators Specially Useful in Chelating Tc99m

A thiolactone diaminedithlol bifunctional chelating agent

p-carboxyethylphenylglyoxal-di-N-methylthioxemicro-bazone

A diamide dimercaptide chelating agent

A hydroxy compound (e.g. cyclohexanol) attached to cysteine

Bisthio semicarbazones

Cyclan

Diamido dithio ligands

Radionuclides

Indium 111

Thallium 201

Technetium 99m

Other Compounds and Chelates Suitable for MRI Imaging

Gadolinium, cadmium, Strontium, Chromium; ferrous

gluconate; manganese; nickel, piperidine and pyrrolidine

NSFR derivatives, ferric ammonium 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 Deaminase; Amylase; Angiotensin I, II, III; Calmodulin; Carboxypeptidase; Catalase; Cellulase; Cholesterol oxidase; Cholinesterase; Chymotrypsin; Collagenase; Complement cascade proteins; Creatine phosphokinase; Deoxyribonuclease I, II; Dipeptidyl peptidase; DNA polymetase; Endoproteinase; Endonucleases; Esterases; beta-Galacatosidase; Galactose oxidase; Galactose dehydrogenase; Glucose dehydrogenase; Glucose oxidase; Glucose-6-phosphate dehydrogenase; Glucuronidase/Aryl sulfatase; Glutamate-oxaloacetate transaminase; Glutamate-pyruvate transaminase; Glutathione reductase; Clutathione perbxidase; Glycopeptidase; Hementin; Hemoglobin; Hexokinase; Hyaluronidase; Lactate dehydrogenase; Lactoperoxidase; Lactamase; Lipase; Myokinase; Neuraminidase; Nicotinamide-adenine Dinucleotide kinase; Nicotinamide-adenine Dinucleotide oxidase; Nuclease; Nucleosidase; Papain; Peroxidase; Phenylalanine dehydrogenase; phosphatase (acid or alkaline); Phosphodiesterase; Phospholipase (A2, C, D); Plasmin; Proteases; Protein Kinase C; Proteinase K; Renin; Reverse transcriptase; Ribonuclease (A, T1, T2, U2); RNA polymerase; Sialytransferase; Streptokinase; Subtilisin A; Superoxide dismutase; Terminal transferase; Urease; Urokinase

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 genes, Herpes Simplex genes, ribosomal RNA, human DNA and RNA; Genes coding for ribozymes; genes coding for antibiotics (e.g., 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 (e.g., 1-O-hexadecyl-2-acetyl-sn-glycero-3-phosphocholine; 3-O-hexadecyl-2-acetyl-sn-glycero-1-phosphocholine; 1-O-hexadecyl-sn-glycero-3-phosphocholine; 1-O-hexadecyl-2-N-methylcarbamyl-sn-glycero-3-phosphocholine; 1-O-hexadecyl-2-thioacetyl-2-deoxy-sn-glycero-3-phosphocholine; 1-O-hexadecyl-2-acetyl-sn-glycero-3-phospho(N-methylpyrrolidino)-ethanolamine); 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-(3,4,5-trimethoxyphenyl); 1-0-Hexadecyl-2-acetyl-sn-glycero-3-phospho(N,N,N-trimethyl)-hexanolamine.

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

Gly-Arg-Gly-Glu-Ser-Pro (inactive control peptide)

Gly-Arg-Gly-Asp-Asn-Pro

n-Methyl-Gly-Arg-Gly-Asp-Ser-Pro

Arg-Gly-Asp-Ser

Gly-Arg-Gly-Asp-Ser

Gly-Arg-Gly-Asp-Ser-Pro-Cys

Gly-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 (chlorambucil, cyclophosphamide, mechlorethamine, melphalan), ethyleneamine derivatives (thiotepa), alkyl sulfonates (busulfan), 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 vinca alkaloids (vinblastine, vincristine, paclitaxel), podophyllotoxin and its derivatives (etoposide); antibiotics (bleomycin, 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)

5. Enzymes, such as asparaginase

6. Miscellaneous agents, such as substituted urea (hydroxyurea), methyl hydrazine derivative (procarbazine), adrenocortical suppressant (mitotane), heavy metal complex (cisplatin, carboplatin)

The 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.

Number	Name	Date	Kind
3565559	Sato et al.	Feb 1971	A
3663685	Evans et al.	May 1972	A
3663686	Grotenhuis et al.	May 1972	A
4022758	Andersson et al.	May 1977	A
4107288	Oppenheim et al.	Aug 1978	A
4115534	Ithakissios	Sep 1978	A
4147767	Yapel, Jr.	Apr 1979	A
4269821	Kreuter et al.	May 1981	A
4325937	Spence et al.	Apr 1982	A
4357259	Senyei et al.	Nov 1982	A
4410507	Chia et al.	Oct 1983	A
4427650	Stroetmann	Jan 1984	A
4492720	Mosier	Jan 1985	A
4619913	Luck et al.	Oct 1986	A
4647536	Mosbach et al.	Mar 1987	A
4818542	Deluca et al.	Apr 1989	A
4822535	Ekman et al.	Apr 1989	A
4921705	Arai et al.	May 1990	A
4925677	Feijen	May 1990	A
4963367	Ecanow	Oct 1990	A
5004202	Hutchinson	Apr 1991	A
5049322	Devissaguet et al.	Sep 1991	A
5069936	Yen	Dec 1991	A
5104674	Chen et al.	Apr 1992	A
5149540	Kunihiro et al.	Sep 1992	A
5206023	Hunziker	Apr 1993	A
5308620	Yen	May 1994	A
5374441	Gibson et al.	Dec 1994	A
5518709	Sutton et al.	May 1996	A
5616311	Yen	Apr 1997	A
5691160	Janmey et al.	Nov 1997	A
5716643	Yen	Feb 1998	A
5725804	Yen	Mar 1998	A
5741478	Osborne et al.	Apr 1998	A
5763416	Bonadio et al.	Jun 1998	A
5955108	Sutton et al.	Sep 1999	A
5977313	Heath et al.	Nov 1999	A
6264988	Yen	Jul 2001	B1

Number	Date	Country
WO 9112823	Sep 1991	WO
WO 9408627	Apr 1994	WO
WO 9618388	Jun 1996	WO
WO 9710850	Mar 1997	WO
WO 9744015	Nov 1997	WO

	Number	Date	Country
Parent	08/554919	Nov 1995	US
Child	08/952765		US
Parent	08/471650	Jun 1995	US
Child	08/554919		US
Parent	08/212546	Mar 1994	US
Child	08/471650		US
Parent	08/069831	Jun 1993	US
Child	08/212546		US
Parent	07/959560	Oct 1992	US
Child	08/069831		US
Parent	07/641720	Jan 1991	US
Child	07/959560		US

Fibrinogen-coated particles for therapeutic use

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Abstract

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CROSS REFERENCE TO RELATED APPLICATIONS

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US Referenced Citations (38)

Foreign Referenced Citations (5)

Non-Patent Literature Citations (10)

Continuation in Parts (6)

Entry
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