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WO1996039128A1 - Particules proteiques a usage therapeutique et diagnostique - Google Patents

Particules proteiques a usage therapeutique et diagnostique Download PDF

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Publication number
WO1996039128A1
WO1996039128A1 PCT/US1996/009458 US9609458W WO9639128A1 WO 1996039128 A1 WO1996039128 A1 WO 1996039128A1 US 9609458 W US9609458 W US 9609458W WO 9639128 A1 WO9639128 A1 WO 9639128A1
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WIPO (PCT)
Prior art keywords
particles
accordance
suspension
gly
arg
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PCT/US1996/009458
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English (en)
Inventor
Richard C. K. Yen
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Hemosphere, Inc.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from US08/471,650 external-priority patent/US5725804A/en
Application filed by Hemosphere, Inc. filed Critical Hemosphere, Inc.
Priority to AU61002/96A priority Critical patent/AU6100296A/en
Priority to US08/952,765 priority patent/US6391343B1/en
Priority to EP96918313A priority patent/EP0831793A4/fr
Priority to JP9501772A priority patent/JPH11507630A/ja
Publication of WO1996039128A1 publication Critical patent/WO1996039128A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51—Nanocapsules; Nanoparticles
    • A61K9/5107—Excipients; Inactive ingredients
    • A61K9/513—Organic macromolecular compounds; Dendrimers
    • A61K9/5169—Proteins, e.g. albumin, gelatin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6921—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere
    • A61K47/6927—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/16—Agglomerates; Granulates; Microbeadlets ; Microspheres; Pellets; Solid products obtained by spray drying, spray freeze drying, spray congealing,(multiple) emulsion solvent evaporation or extraction
    • A61K9/1605—Excipients; Inactive ingredients
    • A61K9/1611—Inorganic compounds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/16—Agglomerates; Granulates; Microbeadlets ; Microspheres; Pellets; Solid products obtained by spray drying, spray freeze drying, spray congealing,(multiple) emulsion solvent evaporation or extraction
    • A61K9/1605—Excipients; Inactive ingredients
    • A61K9/1617—Organic compounds, e.g. phospholipids, fats
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/16—Agglomerates; Granulates; Microbeadlets ; Microspheres; Pellets; Solid products obtained by spray drying, spray freeze drying, spray congealing,(multiple) emulsion solvent evaporation or extraction
    • A61K9/1605—Excipients; Inactive ingredients
    • A61K9/1629—Organic macromolecular compounds
    • A61K9/1658—Proteins, e.g. albumin, gelatin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/04—Antihaemorrhagics; Procoagulants; Haemostatic agents; Antifibrinolytic agents
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y5/00—Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery

Definitions

  • 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 eguilibration 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.
  • 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 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.
  • RES reticuloendothelial system
  • Tiny air bubbles 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.
  • 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.
  • 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 biologies such as oxygen, enzymes, drugs, and information molecules (DNA, RNA and hybrid molecules of DNA and RNA) to cells and tissues.
  • nutrients and biologies such as oxygen, enzymes, drugs, and information molecules (DNA, RNA and hybrid molecules of DNA and RNA)
  • information molecules DNA, RNA and hybrid molecules of DNA and RNA
  • a variety of methods have been used during or after synthesis to prevent resolubilization of the protein particles. These methods include heat denaturization (see, Evans, et al . U.S. Patent No. 3,663,685 and Widder, et al . , Adv. Pharmacol , and Chemother.
  • United States Patent No. 4,269,821, Kreuter, et al . , May 26, 1981, for "Biological Material” discloses processes for the preparation of submicroscopic particles of a physiologically acceptable polymer associated with a biologically active material by using a cross-linking agent such as a polymerisable material soluble in a liquid medium (methyl methacrylate as an example) .
  • United States Patent No. 3,663,685, Evans et al . , May 16, 1972, for "Biodegradable Radioactive Particles” (hereafter "Evans") discloses a method of preparing biodegradable radioactive particles by using heated water-oil solutions. Widder, et al .
  • 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.
  • 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.
  • 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.
  • the suspension may be diluted in an alcohol-free aqueous medium to lower the alcohol content.
  • the suspension may be dialyzed against an aqueous medium to remove any molecular species small enough to pass through the dialysis membrane (i.e.
  • 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.
  • 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.
  • 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.
  • the protein particles which are the subject of this invention are monodisperse particles, generally spherical in shape.
  • monodisperse 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).
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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, B-propanol, isopropanol or ⁇ -butanol. Among these alcohols, ethanol and n-butanol are particularly preferred.
  • 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.
  • 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.
  • 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.
  • HSA human serum albumin
  • SLS sodium lauryl sulfate
  • 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.
  • Tube Contents (balance: water to achieve total volume of 1.0 mL before addition of alcohol) Size (and
  • This example illustrates the use of a stabilizing agent to prevent resolubilization of human serum albumin (HSA) spheres.
  • HSA human serum albumin
  • 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.
  • the HSA concentration is 50 mg/mL or lower, the spheres or particles formed are typically 0.2 to 0.5 microns in diameter.
  • 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.
  • This example illustrates particular agents that were not effective in protecting spheres against resolubilization.
  • NaN0 2 sodium 10 10,000 nitrite
  • 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 ⁇ L 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.
  • EXAMPLE 5 This example illustrates that premixing a stabilizing agent with HSA solution before the addition of alcohol results in stable spheres.
  • 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 ⁇ L 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.
  • This example illustrates that protein particles can be stabilized by a hydrogen-accepting agent in the presence of a hydrogen-donating agent.
  • 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 ⁇ L 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.
  • NADP hydrogen-accepting agent
  • NADPH hydrogen-donating agent
  • 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 ⁇ L 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.
  • 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.
  • a stabilizing agent To demonstrate that enzymes trapped within the interior and on the surface of protein particles stabilized with a stabilizing agent can retain their catalytic function, 5 ⁇ L 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 ⁇ L 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.
  • 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 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 conjuggated 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.
  • 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 9 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.
  • an antibody a commercial source of polyclonal goat anti-human fibrinogen IgG was used.
  • the suspension of antibody-containing spheres (1.0 L) was incubated overnight with sodium bisulfite (5 ⁇ L 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) .
  • 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.
  • a sheep anti-mouse IgG conjuggated 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.
  • 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.
  • 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 ⁇ g 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 ⁇ L of sodium bisulfite (0.1 M) per mL suspension.
  • the spheres were then washed in normal saline (3 x 10 mL) and digested in trypsin to release any DNA previously incorporated inside or on the surface of spheres. Detection of DNA was performed by the Intvogen DNA DipStick Kit, which showed strong positivity with the trypsin digest of spheres prepared from DNA-containing HSA solutions, but not from control spheres or the supernatants of both experimental and control sphere suspensions.
  • Example 12 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).
  • the stabilizing agent sodium bisulfite 0.1 M
  • 5 ⁇ L of a suspension of DNA or RNA was added to the protein spheres to result in a final concentration of 5 ⁇ g DNA/RNA per mL of protein suspension.
  • 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 lmM solution of the potential stabilizing agent was added to 2 L of sphere suspensions. Only two final concentrations (lOmM 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.
  • 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 chloiride magnesium chloride manganese chloride ferric nitrate Vitamins: cyanobalamin (B 12 ) ergocalcifero (D 2 ) Acids: folinic acid
  • N-acetylneuraminic acid w-acetamido-2-deoxy-D-glucose
  • Amines ethanolamine Others: choline bitartrae cephalothin glycerol heparin chear germ agglutinin (lectin)
  • 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.
  • 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.
  • 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,
  • the final Na bisulfite concentration in all three methods will be about ImM.
  • the spheres had to be incubated in the stabilizing agent for more than 30 minutes to stabilize the spheres.
  • 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.
  • 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.
  • 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.
  • fibrinogen-coated particles in co- aggregation with platelets or in improving bleeding time.
  • Fibrinogen on the surface of platelets is further converted into fibrin by the action of thrombin.
  • 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.
  • DIC disminated intravascular coagulation
  • 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.
  • 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.
  • 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.
  • biocompatible is employed in its conventional sense, that is, to denote compounds which do not substantially interfere or interact with the tissues, fluids and other components of the body in an adverse fashion in the particular application of interest.
  • These matrices include microspheres made from natural and synthetic polymers, phospholipid vesicles (see U.S. Patent No.
  • polyesters and/or polyamides encapsulated in methylcellulose U.S. Patent No. 5,233,995
  • gelatin gelatin
  • albumin collagen
  • collagen U.S. Patent No. 4,844,882
  • aliphatic and alicyclic carbon-containing compounds U.S. Patent No. 5,143,716
  • polya inoacids U.S. Patent No. 4,247,406
  • polyglutamate e.g., polyglutamate
  • pblylysine either separately or in combination with proteinaceous microspheres
  • albumin EP 0 633 030 WO 92/17213
  • WO 91/12823 synthetic polymers WO 92/17514, U.S.
  • Thrombospheres 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.
  • Severely thrombocytopenic rabbits received i.v., either TS, 7.5xl0 9 /kg; control albumin spheres (CS) which had no fibrinogen, 7.5xl0 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.
  • TS 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.
  • 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.
  • fibrinogen on the surface of the particles remains undigested and therefore unreactive.
  • 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.
  • 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.
  • biocompatible matrices are illustrative but not limiting: cellulose agarose hemicellulose starch (amylose, amylopectin) mannans glucans xanthans pullutans arabinans arabinogalactans arabinoglucans arbinoglucoronomannans xylans arabinoxylans ⁇ arrageenans 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:
  • 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.
  • a competing small molecule such as glycine, for example, competing for the aldehyde site
  • treatment or “treating” of a condition and/or a disease in a mammal, means:
  • 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.
  • 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 ,01%w to 99.99%w excipient.
  • the drug is present at a level of about 10%w to about 70%w.
  • 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.
  • 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.
  • 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.
  • 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.
  • human interleukin-2 recombinant 13. anti-human platelet-derived growth factor beta receptor
  • alpha 2-macroglobulin 16. streptokinase
  • HIV-1 protease substrate acetyl-ser-gln-asn-tyr-pro-val-val-amide
  • Tc99m 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.
  • 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.
  • 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.
  • 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 (Tc0 4 ⁇ ) to Tc0 2 ⁇ , which then binds to the sulhydryl group on the protein molecules, or to the sites on the chelating agents designed to bind the Tc0 2 ⁇ . It is expected that more Tc99m binds to particles through chelating agents than without chelating agents.
  • 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 Tc0 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.
  • reducing agents are dithiothreitol, dithioerythritol, ascorbic acid, 2-mercaptoethanol, and pyrophosphate.
  • the reduced Tc0 2 ⁇ may first be stabilized by an intermediate product involving D-glucarate.
  • 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.
  • biologically active molecules 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.
  • subunits or fragments of these molecules 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.
  • 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, hentriacont
  • 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
  • Doxyl Nitroxides e.g., 3-beta-doxyl-5-alpha-cholestane; Proxy1 Nitroxides, e.g., 3-(4-nitrophenoxycarbonyl)-proxyl; Tempo Nitroxides, e.g., Tempo; DL-t-Butyl Nitroxide;
  • HETEs 5(S)-HETE[5(S) -hydroxy-6-trans-8-cis-ll-cis-14-cis- eicosatetraenoic acid]; 11(S) -HETE[11(S)-hydroxy-5- cis-8-cis-l2-trans-l4-cis-eicosatetraenoic acid] ;12(R)-HETE[12(R)-hydroxy-5-cis-8-cis-10-trans-trans-
  • DiHETEs 5(S) ,6(R)-DiHETE[5(S) ,6(R)-dihydroxy-7-trans-9- trans-ll-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-ll-cis-13- trans-eicosatetraenoic acid] Other arachidonic acid cascade related compounds: 13-Azaprostanoic acid; Baicalein; 7-7- dimethyleicosadienoic acid; 5,8, lleicosatriynoic acid; 5,8,11,14-eicosatetraynoic acid; oleoyloxyethyl
  • Phosphocholine Phosphocholine; sodium furegrelate; w-3 fatty acids; leukotrienes (LTA4, LTB4, LTC4, LTD4, LTE4) ; lipoxin (A4, B4) , Prostaglandins (A2, B2, D2, El, E2, F2 ⁇ , 12, G2, H2) ; 16-16-Dimethyl-prostaglandin E2; 6-Keto-prostaglandin Fl ⁇ ; 2,3-Dinor 6-keto-prostaglandin Fl ⁇ ; 9,ll-Dideoxy-9 ⁇ , ll ⁇ - methanoepoxyprostaglandin-F2c.; 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;
  • Bradykinin Potentiator e.g. 5a, 9a, B, C
  • Fibroblast Growth Factor Acidic fragment 1-11 Fibroblast Growth Factor, Basic fragment 1-24
  • Fibronectin Related Peptide Fibronectin Pepsin e.g. 50K
  • Fibronectin Chymotrypsin e.g. 4OK, 45K, 12OK
  • Fibronectin Trypsin (e.g. 30K, 60 K)
  • GRF 1-40 Human Growth Hormone Release Inhibiting Factor
  • Hepatitis A viral proteins and peptides Hepatitis B Virus Pre-S Region 120-145)
  • Insulin Chain B oxidized
  • 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-2 Receptor C-Terminal Sequence Interleukin (e.g. l alpha, 2, 6, gamma)
  • Beta-Melanocyte Stimulating Hormone beta-Melanocyte Stimulating Hormone
  • Neurokinin e .g. , A, Nle-10, B, MePhe7-B
  • Neuromedins e . g. , B,C
  • Octadecaneuropeptide e.g. 6, 1 , 8
  • Platelet Derived Growth Factor (AB-chain, heterodimer, AA homodimer, BB homodimer)
  • Protein G (binds to Fc region, specially of IgGl subclass)
  • Tumor Necrosis factor e.g. alpha
  • Vasoactive Intestinal contractor Vasoactive Intestinal Peptide and analogs Vasopressin and analogs Vasotocin Versican Vitronectin Xenopsin Yeast Alpha-Factor
  • Carboxyethylthioethyl 2-Acetamido-2-deoxy-3-0-Beta-D- galactopyranosyl-beta-D-glucopyranoside-HSA Conjugate Carboxyethylthioethyl 2-Acetamido-2-deoxy-4-0-beta-D- galactopyranosyl-beta-D-glucopyranoside-HSA Conjugate
  • Glucopyranosy1-alpha-D-glucopyranosy1)-alpha-D- glucopyranosyl)-beta-D-glucopyranoside-HSA Conjugate Free oligosaccharides and simple derivatives:
  • Carbomethoxyethylthioethyl 2-Acetamido-2-deoxy-4-0-beta-D- galactopyranosyl-beta-D-glucopyranoside Carbomethoxyethylthioethyl 4-0-(4-0-(6-0-alpha-D- Glucopyranosyl-alpha-D-glucopyranosyl)-alpha-D- glucopyranosyl)-beta-D-glucopyranoside p-Nitrophenyl 2-Acetamido-2-deoxy-3-0-(2-acetamido-2- deoxy-bcta-D-glucopyranosy1)-alpha-D- galactopyranoside p-Nitrophenyl 2-Acetamido-2-deoxy-3-0-(Beta-D- galactopyranosyl)-alpha-D-galactopyranoside p-Nitrophenyl 2-
  • Octadecylthioethyl 4-0-alpha-D-Galactopyranosyl-beta-D- galactopyranoside Octadecylthioethyl 4-0-(4-0-(6-0-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:
  • Coagulation Factors Coagulation factor Inhibitors
  • Crotalase Ecarin; Factor I, II, III, IV, V; Factor V
  • Factor Factor; Factor IX; Factor X; Factor X, Activiated (Xa) Factor X Activating Enzyme; Factor XI, XII, XIII; Fibrin;
  • Fibrin/Fibrinogen Degradation Products Fibrinogen
  • Platelet Factor 4 Platelet Aggregation Reagent; Brain Cephalin; Snake venoms; Streptokinase; Thrombins;
  • Plasminogen Activator Urokinase Agents and drugs used to treat or prevent HIV infection:
  • 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;
  • 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
  • Humoral Factor Humoral Factor; Thymopentin; Tumor Necrosis Factor; ketoconazole; fluconazole; Eflornithine; Spiramycin;
  • DHPG Ganciclovir
  • 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:
  • Apolipoprotein A (I and II), B, CIII, CII, Cl, E High Density Lipoprotein Low Density Lipoprotein Very Low Density Lipoprotein Lipoprotein Lipase Lipoteichoic Acid Lipoxidase Diaphorase Lipoxin A4, B4 Lipoxygenase
  • Iminodiacetic Acid e.g., dimethyl-ida, paraisopropyl-ida, parabutyl-ida, diisopropyl-ida, diethyl-ida
  • EDTA Ethylenediaminetetraacetic acid
  • NTA Netriloacetic acid
  • TPP Tripolyphosphate Cysteine
  • NITR5, NITR7, DM-nitrophen, NITRS/AM Ammonium N- nitrosophenyl-hydroxylamine; Ammonium purpurate; alpha-Benzoin oxime; N, N-Bis-(hydroxyethyl)-glycine;
  • Phenanthroline Potassium ethyl xanthate; Salicylic acid; sodium diethyldithio-carbamate; 2-Thenoyl-2- furoylmethane; Thenoyl-trifluoro-acetone; Thiourea; Triethylenetetramine Deferoxamine mesylate
  • a thiolactone diaminedithiol bifunctional chelating agent p-carboxyethylphenylglyoxal-di-N-methylthioxemicro-bazone
  • a diamide dimercaptide chelating agent A hydroxy compound (e . g. cyclohexanol) attached to cysteine
  • 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
  • Dipeptidyl peptidase Dipeptidyl peptidase; DNA polymerase; 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; Glutathione peroxidase; Glycopeptidase; Hementin; Hemoglobin; Hexokinase; Hyaluronidase; Lactate dehydrogenase; Lactoperoxidase; Lactamase; Lipase; Myokinase; Neura
  • Platelet related proteins and enzymes Platelet factor 4; 1-3-Dioxolane; l-0-Hexadecyl-2-acetyl-sn-glucero-3-phospho- (N,N,N-trimethyl)-hexanolamine; Platelet activating factors (e .g.
  • l-0-hexadecyl-2-acetyl-sn- glycero-3-phosphocholine 3-0-hexadecyl-2-acetyl-sn- glycero-1-phosphocholine; l-0-hexadecyl-sn-glycero-3- phosphocholine; l-O-hexadecyl-2-N-methylcarbamyl-sn- glycero-3-phosphocholine; l-0-hexadecyl-2-thioacetyl-2- deoxy-sn-glycero-3-phosphocholine; l-O-hexadecyl-2-acetyl- sn-glycero-3-phospho(N-methylpyrrolidino)-ethanolamine) ;
  • Other compounds Protein A,B,C,G,S; Ricin A; Proadifen (SKF-525A)1 Taxol;
  • Alkylating agents such as nitrogen mustards (chlorambucil, cyclophosphamide, mechlorethamine, melphalan) , ethyleneamine derivatives (thiotepa) , alkyl sulfonates (busulfan) , nitrosoureas (carmustine, lomustine) , triazenes (dacarbasine)
  • Antimetabolites such as folic acid analogues (methotrexate) , pyrimidine analogues (cytarabine, floxuridine, fluorouracil) , purine analogues ( ercaptopurine, thioguanine)
  • Natural Products such as vinca alkaloids (vinblastine, vincristine, paclitaxel) , podophyllotoxin and its derivatives (etoposide) ; antibiotics (bleomycin. dactinomycin, doxorubicin, daunomycin, mithramycin, mitomycin)
  • 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, edroxyprogesterone, megestrol) , antiestorgen (tamoxifen)
  • prednisone adrenal corticosteroids
  • estrogens chlorotrianisene, conjugated estrogens, diethylstilbestrol, diethylstilbestrol diphosphate, ethinyl estradiol
  • androgens calusterone, dromostanolone propionate, fluoxymesterone, testolactone, testosterone propionate, testosterone en
  • Enzymes such as asparaginase
  • Miscellaneous agents such as substituted urea (hydroxyurea) , methyl hydrazine derivative (procarbazine) , adrenocortical suppressant (mitotane) , heavy metal complex (cisplatin, carboplatin)

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Abstract

On stabilise vis-à-vis de la resolubilisation, des particules d'albumine de l'ordre du nanomètre et du micromètre dans une suspension aqueuse sans utiliser d'agent de réticulation et sans dénaturation, en incorporant un agent de stabilisation à la composition particulaire. Les agents de stabilisation selon l'invention sont, entre autres, des agents de réduction, des agents d'oxydation, des molécules acceptant l'hydrogène, des polymères de masse molaire élevée et des composés cycliques contenant du soufre. L'invention porte également sur des particules enrobées de fibrinogène, réticulées ou non, et sur leur utilisation en tant qu'agents de co-agrégation avec les plaquettes et eux-mêmes pour réduire le temps de saignement et amplifier l'action de la thrombine.
PCT/US1996/009458 1991-01-15 1996-06-04 Particules proteiques a usage therapeutique et diagnostique WO1996039128A1 (fr)

Priority Applications (4)

Application Number Priority Date Filing Date Title
AU61002/96A AU6100296A (en) 1995-06-06 1996-06-04 Protein particles for therapeutic and diagnostic use
US08/952,765 US6391343B1 (en) 1991-01-15 1996-06-04 Fibrinogen-coated particles for therapeutic use
EP96918313A EP0831793A4 (fr) 1995-06-06 1996-06-04 Particules proteiques a usage therapeutique et diagnostique
JP9501772A JPH11507630A (ja) 1995-06-06 1996-06-04 治療および診断用途のためのタンパク質粒子

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US08/471,650 1995-06-06
US08/471,650 US5725804A (en) 1991-01-15 1995-06-06 Non-crosslinked protein particles for therapeutic and diagnostic use
US55491995A 1995-11-09 1995-11-09
US08/554,919 1995-11-09

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EP0860167A1 (fr) * 1997-01-30 1998-08-26 Robert Gurny Agents actifs dérivés de l'ADN et de l'ARN inclus dans des nanoparticules
US5977313A (en) * 1996-10-10 1999-11-02 Quadrant Healthcare Limited Platelet substitutes and conjugation methods suitable for their preparation
US6264988B1 (en) 1997-06-05 2001-07-24 Hemosphere, Inc. Fibrinogen-coated microspheres
US7001989B2 (en) 1997-11-14 2006-02-21 Elan Drug Delivery Ltd. Conjugates comprising two active agents
WO2006066595A3 (fr) * 2004-12-22 2006-08-31 Novozymes As Production par recombinaison de serum-albumine
WO2007104422A2 (fr) 2006-03-14 2007-09-20 Lts Lohmann Therapie-Systeme Ag Nanoparticules chargées d'agent actif, à base de protéines hydrophiles
JP2011026330A (ja) * 1999-06-29 2011-02-10 Mannkind Corp ペプチドおよびタンパク質の薬学的因子の精製および安定化
US8080516B2 (en) 1999-05-17 2011-12-20 Conjuchem, Llc Long lasting synthetic exendin-4 peptide conjugates
EP2753349A4 (fr) * 2011-09-10 2015-05-13 Richard C K Yen Particules sous-micrométriques pour diminuer la transfusion
CN105085611A (zh) * 2011-11-18 2015-11-25 A&Pep公司 具有皮肤美白效果的烟酸-肽和其用途
US9339584B2 (en) 2006-11-27 2016-05-17 Haemostatix Limited Biogel
EP3272335A1 (fr) * 2016-07-20 2018-01-24 Richard C.K. Yen Préparations de nanosphères d'albumine pour réguler le saignement d'opérations chirurgicales
CN112236440A (zh) * 2018-05-28 2021-01-15 江阴贝瑞森制药有限公司 新的药物用途
US11059859B2 (en) 2017-07-05 2021-07-13 Jiangyin Usun Pharmaceutical Co., Ltd. Anti-inflammatory use of peptide
CN113797861A (zh) * 2021-09-16 2021-12-17 江苏月旭新材料科技有限公司 一种羟丙基葡聚糖微球及其制备方法和用途
US20240124521A1 (en) * 2019-09-14 2024-04-18 Enlitisa (Shanghai) Pharmaceutical Co., Ltd. New peptides
WO2024169972A1 (fr) 2023-02-13 2024-08-22 Enlitisa (Shanghai) Pharmaceutical Co., Ltd. Nouveaux oligopeptides multifonctionnels
WO2024260465A1 (fr) 2023-06-22 2024-12-26 Enlitisa (Shanghai) Pharmaceutical Co., Ltd. Nouveaux oligopeptides multifonctionnels

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US4410507A (en) * 1981-08-28 1983-10-18 Solco Basel Ag Process for the preparation of physiologically degradable, colloidal radioisotope carriers and their use
US4818542A (en) * 1983-11-14 1989-04-04 The University Of Kentucky Research Foundation Porous microspheres for drug delivery and methods for making same
US5104674A (en) * 1983-12-30 1992-04-14 Kraft General Foods, Inc. Microfragmented ionic polysaccharide/protein complex dispersions
US4822535A (en) * 1985-07-12 1989-04-18 Norsk Hydro A.S. Method for producing small, spherical polymer particles
US5069936A (en) * 1987-06-25 1991-12-03 Yen Richard C K Manufacturing protein microspheres
US4921705A (en) * 1987-07-14 1990-05-01 Nippon Oil And Fats Co., Ltd. Lipid powder having cross-linked coating thereon and process for preparing same
US5149540A (en) * 1990-03-01 1992-09-22 Mochida Pharmaceutical Co., Ltd. Thrombin composition for oral administration
US5374441A (en) * 1992-02-04 1994-12-20 Rutgers, The State University Of New Jersey Heat stable fat substitute compositions and process

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Cited By (28)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5977313A (en) * 1996-10-10 1999-11-02 Quadrant Healthcare Limited Platelet substitutes and conjugation methods suitable for their preparation
JP2001504813A (ja) * 1996-10-21 2001-04-10 クウォドラント、ヘルスケアー、（ユーケー）、リミテッド 代用血小板、及びそれらの製造に適した抱合法
EP0860167A1 (fr) * 1997-01-30 1998-08-26 Robert Gurny Agents actifs dérivés de l'ADN et de l'ARN inclus dans des nanoparticules
WO1998033478A3 (fr) * 1997-01-30 1998-12-10 Robert Gurny Agents actifs a base d'arn et d'adn dans des nanoparticules
US6264988B1 (en) 1997-06-05 2001-07-24 Hemosphere, Inc. Fibrinogen-coated microspheres
EP1003481A4 (fr) * 1997-06-05 2002-10-23 Hemosphere Inc Microspheres enrobees de fibrinogene
US7001989B2 (en) 1997-11-14 2006-02-21 Elan Drug Delivery Ltd. Conjugates comprising two active agents
US8080516B2 (en) 1999-05-17 2011-12-20 Conjuchem, Llc Long lasting synthetic exendin-4 peptide conjugates
US8084414B2 (en) 1999-05-17 2011-12-27 Conjuchem, Llc Methods involving long lasting synthetic exendin-4-peptide conjugates
JP2011026330A (ja) * 1999-06-29 2011-02-10 Mannkind Corp ペプチドおよびタンパク質の薬学的因子の精製および安定化
EP2169067A1 (fr) * 2004-12-22 2010-03-31 Novozymes A/S Production de sérum-albumine par recombinaison
WO2006066595A3 (fr) * 2004-12-22 2006-08-31 Novozymes As Production par recombinaison de serum-albumine
WO2007104422A2 (fr) 2006-03-14 2007-09-20 Lts Lohmann Therapie-Systeme Ag Nanoparticules chargées d'agent actif, à base de protéines hydrophiles
WO2007104422A3 (fr) * 2006-03-14 2008-03-20 Lohmann Therapie Syst Lts Nanoparticules chargées d'agent actif, à base de protéines hydrophiles
US9339584B2 (en) 2006-11-27 2016-05-17 Haemostatix Limited Biogel
US9913876B2 (en) 2006-11-27 2018-03-13 Haemostatix Limited Biogel
EP2753349A4 (fr) * 2011-09-10 2015-05-13 Richard C K Yen Particules sous-micrométriques pour diminuer la transfusion
CN105085611A (zh) * 2011-11-18 2015-11-25 A&Pep公司 具有皮肤美白效果的烟酸-肽和其用途
CN105085611B (zh) * 2011-11-18 2019-07-19 A&Pep公司 具有皮肤美白效果的烟酸-肽和其用途
EP3272335A1 (fr) * 2016-07-20 2018-01-24 Richard C.K. Yen Préparations de nanosphères d'albumine pour réguler le saignement d'opérations chirurgicales
US11059859B2 (en) 2017-07-05 2021-07-13 Jiangyin Usun Pharmaceutical Co., Ltd. Anti-inflammatory use of peptide
US12129313B2 (en) 2017-07-05 2024-10-29 Enlitisa (Shanghai) Pharmaceutical Co., Ltd. Anti-inflammatory use of peptide
CN112236440A (zh) * 2018-05-28 2021-01-15 江阴贝瑞森制药有限公司 新的药物用途
US20240124521A1 (en) * 2019-09-14 2024-04-18 Enlitisa (Shanghai) Pharmaceutical Co., Ltd. New peptides
CN113797861A (zh) * 2021-09-16 2021-12-17 江苏月旭新材料科技有限公司 一种羟丙基葡聚糖微球及其制备方法和用途
CN113797861B (zh) * 2021-09-16 2024-03-29 江苏月旭新材料科技有限公司 一种羟丙基葡聚糖微球及其制备方法和用途
WO2024169972A1 (fr) 2023-02-13 2024-08-22 Enlitisa (Shanghai) Pharmaceutical Co., Ltd. Nouveaux oligopeptides multifonctionnels
WO2024260465A1 (fr) 2023-06-22 2024-12-26 Enlitisa (Shanghai) Pharmaceutical Co., Ltd. Nouveaux oligopeptides multifonctionnels

Also Published As

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CA2220895A1 (fr) 1996-12-12
JPH11507630A (ja) 1999-07-06
EP0831793A1 (fr) 1998-04-01
EP0831793A4 (fr) 2000-11-15
AU6100296A (en) 1996-12-24

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