Search Images Maps Play YouTube News Gmail Drive More »
Sign in
Screen reader users: click this link for accessible mode. Accessible mode has the same essential features but works better with your reader.

Patents

  1. Advanced Patent Search
Publication numberUS20020130430 A1
Publication typeApplication
Application numberUS 09/750,473
Publication dateSep 19, 2002
Filing dateDec 29, 2000
Priority dateDec 29, 2000
Also published asUS7147806, US20060033224
Publication number09750473, 750473, US 2002/0130430 A1, US 2002/130430 A1, US 20020130430 A1, US 20020130430A1, US 2002130430 A1, US 2002130430A1, US-A1-20020130430, US-A1-2002130430, US2002/0130430A1, US2002/130430A1, US20020130430 A1, US20020130430A1, US2002130430 A1, US2002130430A1
InventorsTrevor Castor
Original AssigneeCastor Trevor Percival
Export CitationBiBTeX, EndNote, RefMan
External Links: USPTO, USPTO Assignment, Espacenet
Methods for making polymer microspheres/nanospheres and encapsulating therapeutic proteins and other products
US 20020130430 A1
Abstract
This invention is for an improved process to formulate polymeric microspheres/nanospheres and encapsulate therapeutic proteins and other useful substances. Non-toxic supercritical or near-critical fluids with/without polar cosolvents are utilized to solubilize biodegradable polymers and form uniform polymer microspheres and nanospheres to encapsulate proteins with controlled-release characteristics.
Images(4)
Previous page
Next page
Claims(20)
What is claimed is:
1. A method of making polymeric spheres having an average diameter of between 0.01 and 10.0 microns, comprising the steps of:
a.) providing a polymer solution of a polymeric material dissolved in a first fluid, said first fluid consisting of a supercritical, critical or near-critical fluid; and,
b.) depressurizing said polymer solution as said polymer solution exits one or more orifices in the presence of a low solubility fluid, said low solubility fluid having low volatility and said polymeric material in a concentration which exceeds said solubility of said polymeric material in said low solubility fluid, said polymeric material forming spheres having an average diameter between 0.01 and 10.0 microns and said first fluid removed during depressurization.
2. The method of claim 1 wherein said spheres have an average diameter of between 0.1 to 1.0 microns.
3. The method of claim 1 wherein said polymer solution has a bioactive material, said bioactive material dissolved in said polymer solution or held in said polymer solution as a suspension or emulsion.
4. The method of claim 3 wherein said bioactive material is dissolved in said first fluid.
5. The method of claim 3 wherein said bioactive material is held as a suspension in said polymer solution.
6. The method of claim 3 wherein said bioactive material is held as an emulsion in said polymeric solution.
7. The method of claim 3 wherein said bioactive material is dissolved in a second fluid and said second is combined with said first fluid and polymeric material.
8. The method of claim 7 wherein said second fluid is a supercritical, critical or near-critical fluid.
9. The method of claim 7 wherein said bioactive material is held as a suspension in said second fluid.
10. The method of claim 3 wherein said bioactive material is held as an emulsion in said second fluid.
11. The method of claim 1 wherein said polymer solution is depressurized to ambient pressure.
12. The method of claim 1 wherein said low solubility fluid is selected from the group of solvents consisting of PVA, PBS, and liquid nitrogen.
13. The method of claim 1 wherein said polymer is selected from one or more of the group of polymers consisting of poly(L-lactic acid), poly(D, L-lactic acid), poly(glycolic acid) and carboxylic acid and ester derivatives thereof, poly(fumaric anhydride) and poly(sebacic anhydride) and derivatives thereof.
14. A method of making polymeric spheres having an average diameter of between 0.01 and 10.0 microns, comprising the steps of:
a.) providing a polymer solution of a polymeric material in a first fluid, said first fluid consisting of a supercritical, critical or near-critical fluid;
b.) providing a second solution of a bioactive material in a second fluid;
c.) forming an admixture of said first solution and said second solution to form a third solution, said third solution comprising a supercritical, critical or near-critical fluid;
d.) depressurizing said third solution as said third solution exits one or more orifices in the presence of a low solubility fluid, said low solubility fluid having low volatility and said polymeric material in a concentration which exceeds said solubility of said polymeric material in said low solubility fluid, said polymeric material forming spheres having an average diameter of 0.01 to 10.0 microns, said spheres containing said bioactive material and said first fluid removed during depressurization.
15. The method of claim 14 wherein said spheres have an average diameter of between 0.1 to 1.0 microns.
16. An apparatus for forming one or more polymeric spheres having an average diameter of between 0.01 and 10.0 microns, comprising:
a.) an admixture vessel for receiving a polymer solution which polymer solution is a polymeric material dissolved in a first fluid, said first fluid consisting of a supercritical, critical or near-critical fluid;
b.) an orifice nozzle in communication with said admixture vessel for receiving said polymer solution; and,
c.) a depressurization vessel containing a low solubility fluid in communication with said orifice nozzle for receiving a stream of polymer solution as said polymer solution exits one or more orifices in the presence of said low solubility fluid, said low solubility fluid having low volatility and said polymeric material in a concentration which exceeds said solubility of said polymeric material in said low solubility fluid, said polymeric material forming spheres having an average diameter of 0.01 to 10.0 microns and said first fluid removed during depressurization.
17. The method of claim 16 wherein said spheres have an average diameter of between 0.1 to 1.0 microns.
18. The apparatus of claim 16 wherein said admixture vessel receives a bioactive material, said bioactive material dissolved in a solvent or held as a suspension in a fluid or held in an emulsion, and said bioactive material incorporated into said spheres during depressurization.
19. The apparatus of claim 16 wherein said apparatus further comprises a polymer vessel for forming a solution of a polymer in a supercritical, critical or near critical fluid, said polymer vessel in fluid communication with said admixture vessel.
20. The apparatus of claim 16 wherein said apparatus further comprises a bioactive material vessel for forming a suspension, solution or emulsion of said bioactive material in said polymer solution, said bioactive vessel in communication with said admixture vessel.
Description
    FIELD OF THE INVENTION
  • [0001]
    This invention relates to methods for making polymer microspheres and nanospheres and encapsulating therapeutic proteins and other products. The methods relate to improving the drug delivery of therapeutics and other products, and to making therapeutic proteins and other products more orally bioavailable. The methods feature supercritical, critical and near-critical fluids with and without polar cosolvents.
  • BACKGROUND OF THE INVENTION
  • [0002]
    Aspects of the present invention employ materials known as supercritical, critical or near-critical fluids. A material becomes a critical fluid at conditions which equal its critical temperature and critical pressure. A material becomes a supercritical fluid at conditions which equal or exceed both its critical temperature and critical pressure. The parameters of critical temperature and critical pressure are intrinsic thermodynamic properties of all sufficiently stable pure compounds and mixtures. Carbon dioxide, for example, becomes a supercritical fluid at conditions which equal or exceed its critical temperature of 31.1° C. and its critical pressure of 72.8 atm (1,070 psig). In the supercritical fluid region, normally gaseous substances such as carbon dioxide become dense phase fluids which have been observed to exhibit greatly enhanced solvating power. At a pressure of 3,000 psig (204 atm) and a temperature of 40° C., carbon dioxide has a density of approximately 0.8 g/cc and behaves much like a nonpolar organic solvent, having a dipole moment of zero debyes.
  • [0003]
    A supercritical fluid displays a wide spectrum of solvation power as its density is strongly dependent upon temperature and pressure. Temperature changes of tens of degrees or pressure changes by tens of atmospheres can change a compound's solubility in a supercritical fluid by an order of magnitude or more. This feature allows for the fine-tuning of solvation power and the fractionation of mixed solutes. The selectivity of nonpolar supercritical fluid solvents can also be enhanced by addition of compounds known as modifiers (also referred to as entrainers or cosolvents). These modifiers are typically somewhat polar organic solvents such as acetone, ethanol, methanol, methylene chloride or ethyl acetate. Varying the proportion of modifier allows a wide latitude in the variation of solvent power.
  • [0004]
    In addition to their unique solubilization characteristics, supercritical fluids possess other physicochemical properties which add to their attractiveness as solvents. They can exhibit liquid-like density yet still retain gas-like properties of high diffusivity and low viscosity. The latter increases mass transfer rates, significantly reducing processing times. Additionally, the ultra-low surface tension of supercritical fluids allows facile penetration into microporous materials, increasing extraction efficiency and overall yields.
  • [0005]
    A material at conditions that border its supercritical state will have properties that are similar to those of the substance in the supercritical state. These so-called “near-critical” fluids are also useful for the practice of this invention. For the purposes of this invention, a near-critical fluid is defined as a fluid which is (a) at a temperature between its critical temperature (Tc) and 75% of its critical temperature and at a pressure at least 75% of its critical pressure, or (b) at a pressure between its critical pressure (Pc) and 75% of its critical pressure and at a temperature at least 75% of its critical temperature. Inn this definition, pressure and temperature are defined on absolute scales, e.g., Kelvin and psia. Table 1 shows how these requirements relate to some of the fluids relevant to this invention. To simplify the terminology, materials which are utilized under conditions which are supercritical, near-critical, or exactly at their critical point will jointly be referred to as “SCCNC” fluids.
    TABLE 1
    Physical Properties of Critical Fluid Solvents
    Pvap
    BP (psia @ Tc Pc 0.75Tc 0.75Pc
    Fluid Formula (° C.) 25° C.) (° C.) (psia) (° C.) (psia)
    Carbon CO2 −78.5 860 31.1 1070 −45.0 803
    dioxide
    Nitrous N2O −88.5 700 36.5 1051 −41.0 788
    oxide
    Propane C3H8 −42.1 130 96.7 616 4.2 462
    Ethane C2H6 −88.7 570 32.3 709 −44.1 531
    Ethyl- C2H4 −103.8 NA 9.3 731 −61.4 548
    ene
    Freon 11 CCl3F 23.8 15 198.1 639 80.3 480
    Freon 21 CHCl2F 8.9 24 178.5 750 65.6 562
    Freon 22 CHClF2 −40.8 140 96.1 722 3.8 541
    Freon 23 CHF3 −82.2 630 26.1 700 −48.7 525
  • [0006]
    Conventional methods of drug delivery such as tablets or injections provide an initial spike of therapeutic agent in the patient's system followed by a period of decay. Dosage is frequently limited by adverse side effects engendered by the elevated, albeit temporary, high level of agent. Furthermore, as the agent is cleared from the body, its concentration will most likely fall below a useful level prior to the next treatment. For many drugs, the ideal is a steady level over a prolonged period ranging from hours to years. This type of profile may be attained with the use of controlled release technology. Improved techniques for controlled release of therapeutic agents is an area of great importance to the medical field, the pharmaceutical industry, and the public that they serve.
  • [0007]
    One of the most promising methods for controlled release involves the use of degradable or erodable polymers. These are typically formulated as microparticles or microspheres with a size ranging from a maximum of 50 μm down to approximately 0.1 μm. Following administration via ingestion or injection, the polymer is slowly eroded by body fluids to yield biocompatible breakdown products. Concurrently, drug is released from the particle by diffusion through the polymer matrix as well as by surface erosion.
  • [0008]
    The most commonly used bioerodable polymers are of the poly(hydroxyacid) type, in particular poly(L-lactic acid), poly(D,L-lactic acid), poly(glycolic acid), and copolymers thereof. A typical copolymer used for microsphere/microparticle formation is poly(lactide-co-glycolide), abbreviated as PLGA. These materials are broken down in the body to the non-toxic products lactic acid and glycolic acid, and have been approved by the Food and Drug Administration for use as resorbable sutures, in bone implants, and as controlled release microspheres. Other polymers being utilized include poly(funimaric anhydride) and poly(sebacic anhydride). Mathiowitz, E., Jacob, J. S., Jong, Y. S., Carino, G. P., Chickering, D. E., Chaturvedi, P., Santos, C. A., Vijayaraghavan, K., Montgomery, S., Bassett, M. and Morrell, C., Biologically Erodable Microspheres as Potential Oral Drug Delivery Systems, Nature, 386:410-414, 1997. The use of polymeric microspheres for controlled drug delivery has been the subject of a number of reviews. Langer, R., Cima, L. G., Tamada, J. A. and Wintermantel, E.: “Future Directions in Biomaterials,” Biomaterials, 11:738-745, 1990.
  • [0009]
    At present, large scale production of polymeric microspheres utilize many processing steps and require large quantities of organic solvents. The process is very time consuming, costly and inefficient. Generally, such polymeric microspheres have a wide dispersion of particle size. Such polymeric spheres tend to have a median size greater than 100 microns in diameter. In addition, the exposure of therapeutic agent to the organic solvent may adversely affect the integrity of the final product. The process steps may also compromise sterility, or do not provide sterility.
  • [0010]
    Embodiments of the present invention address these problems inherent in the prior art with the application of supercritical, critical or near-critical fluids.
  • SUMMARY OF THE INVENTION
  • [0011]
    Embodiments of the present invention are directed to methods of using supercritical fluids for making uniform polymer spheres. The uniformity and integrity of such spheres make such spheres ideal for containing therapeutic proteins and other products. The methods require reduced processing time and preparation costs.
  • [0012]
    One embodiment of the present invention is a method of making polymeric spheres comprising the steps of providing a polymer solution of a polymeric material dissolved in a first fluid. The first fluid consisting of a supercritical, critical or near-critical fluid. Next, the polymer solution is depressurized as said polymer solution exits one or more orifices in the presence of a low solubility fluid. The low solubility fluid has low volatility and the polymeric material is in a concentration which exceeds the solubility of the polymeric material in the low solubility fluid. The polymeric material forms spheres and the first fluid is removed during depressurization.
  • [0013]
    Embodiments of the present invention feature the formation of spheres having an average diameter of between 0.01 and 10.0 microns and, most preferably, 0.1 and 1.0 microns. The narrow range of diameter of the microspheres that can be attained with the present method is unusual and surprising.
  • [0014]
    Embodiments of the present method are used to incorporate bioactive materials in the spheres. Preferably, the polymer solution has a bioactive material, wherein the bioactive material dissolved or held in said polymer solution as a suspension or emulsion. As used herein, the term “bioactive” refers to compositions which cause a change or modification of a living organism in the nature of pharmaceuticals, drugs, toxins, biocides and the like.
  • [0015]
    Preferably, the bioactive material is dissolved in the first fluid or dissolved in, or is held as a suspension or as an emulsion in a further fluid and the further fluid is combined with the first fluid and polymeric material. Preferably, the fluid used to dissolve or hold the bioactive material is a supercritical, critical or near-critical fluid. A preferred fluid is selected from the group of solvents consisting of PVA, PBS, and liquid nitrogen, with or without a cosolvent, such as an alcohol, an aqueous solvent such as distilled water or mixtures of the aforementioned.
  • [0016]
    Preferably, the polymer solution is depressurized to ambient pressure. A preferred polymer is selected from one or more of the group of polymers consisting of poly(L-lactic acid), poly(D, L-lactic acid), poly(glycolic acid) and carboxylic acid and ester derivatives thereof, poly(fumaric nansanhydride) and poly(sebacic anhydride). Preferred first fluids comprise carbon dioxide, nitrous oxide, ethylene, ethane, propane and fluorohydrocarbons. The first fluid may also contain modifiers. Preferred modifiers are methanol, ethanol, propanol, butanol, methylene chloride, ethyl acetate and acetone. A preferred temperature and pressure for a SCCNC comprising carbon dioxide are a temperature in the range of 10 to 60° C. and a pressure in the range of 1,000 to 5,000 psig.
  • [0017]
    The low solubility fluid, preferably, comprises an aqueous solvent, such as distilled water; a cryogenic fluid, such as liquid nitrogen; an organic solvent, such as an alcohol; or a critical, supercritical or near-critical fluid or mixtures of the aforementioned. Preferably, the low solubility fluid has a chemical agent for stabilizing the polymeric spheres, by cross-linking or other means.
  • [0018]
    One preferred method of making polymeric spheres having an average diameter of between 0.1 and 1.0 microns and a bioactive material comprises the steps of providing a polymer solution of a polymeric material in a first fluid, the first fluid consisting of a supercritical, critical or near-critical fluid. Next, the method comprises the step of providing a bioactive fluid having bioactive material. Next, an admixture of the first solution and the bioactive fluid is formed, to form a admixture solution, the admixture solution comprising a supercritical, critical or near-critical fluid. The admixture solution is depressurized as the admixture solution exits one or more orifices in the presence of a low solubility fluid. The low solubility fluid has low volatility and the polymeric material is in a concentration which exceeds its solubility in this fluid. The polymeric material forms spheres having an average diameter of 0.1 to 1.0 microns which spheres contain the bioactive material as the first fluid is removed during depressurization.
  • [0019]
    A further embodiment of the present invention features an apparatus for forming one or more polymeric spheres. The apparatus comprises an admixture vessel, a depressurization chamber and an orifice. The admixture vessel is for receiving and containing a polymer solution of a polymeric material in a first fluid, the first fluid consisting of a supercritical, critical or near-critical fluid. The depressurization chamber contains a low solubility fluid and is in fluid communication with the admixture vessel by the orifice. The depressurization chamber receives the polymer solution as said polymer solution exits the orifices in the presence of a low solubility fluid. The low solubility fluid has low volatility and the polymeric material is in a concentration which exceeds its solubility in such fluid. The polymeric material forms spheres and the first fluid is removed during depressurization.
  • [0020]
    The apparatus is used to make spheres having an average diameter of 0.01 to 10.0 microns and, most preferably, 0.1 to 1.0 microns.
  • [0021]
    Preferably, the admixture vessel receives a bioactive material. The bioactive material is dissolved in a solvent or held as a suspension in a fluid or held in an emulsion. Such bioactive material is incorporated into the spheres during depressurization.
  • [0022]
    Preferably, the apparatus further comprises a polymer vessel for forming a solution of a polymer in a supercritical, critical or near-critical fluid. The polymer vessel is in fluid communication with the admixture vessel.
  • [0023]
    Preferably, the apparatus further comprises a bioactive material vessel for forming a suspension, solution or emulsion of said bioactive material in a fluid. The bioactive vessel is in communication with the admixture vessel.
  • [0024]
    Sterile filtration and solvent evaporation can then be used to harvest the polymeric spheres. SCCNC fluids also sterilize the materials in which such fluids are incorporated upon rapid depressurization, such as depressurization through an orifice.
  • [0025]
    Surprisingly and unexpectedly, uniform and stable polymeric spheres containing bioactive compositions, such as therapeutic proteins and drugs other products, are formed. The use of SCCNC fluids allows for easy removal of much of the solvent by mere depressurization. Use of a single apparatus to perform polymer sphere formation and encapsulate therapeutic proteins and other products minimizes labor and increases efficiency. Indeed, the entire process can be readily automated. The use of SCCNC fluids allows process conditions to be readily varied by temperature, pressure, or modifier solvents, minimizing equipment needs, processing time, potential for contamination, and loss of yield. The use of SCCNC fluids eliminates the need for toxic organic solvents such as in methylene chloride, ethyl acetate or DMSO and eliminates the presence of these toxic organic solvents in the final product.
  • [0026]
    These and other features and advantages will be readily apparent from the drawing and detailed discussion which follow.
  • BRIEF DESCRIPTION OF THE DRAWINGS
  • [0027]
    [0027]FIG. 1 depicts in schematic form an apparatus embodying features of the present invention;
  • [0028]
    [0028]FIG. 2 shows the in vitro time release characteristics of cytochrome-C from SCCNC polymer spheres; and,
  • [0029]
    [0029]FIG. 3 shows the in vitro time release characteristics of insulin from SCCNC polymer nanospheres.
  • DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS
  • [0030]
    The present method an apparatus will be described with respect to FIG. 1 which depicts in schematic form a polymer sphere apparatus, generally designated by the numeral 11. The polymer sphere apparatus is comprised of the following major elements: a polymer vessel 13, a bioactive injection assembly 15, an admixture chamber 17, a depressurization vessel 19, and an orifice nozzle 21.
  • [0031]
    Polymer vessel 13 is in fluid communication with a SCCNC syringe pump 25 via conduits 27 a and 27 b. SCNCC pump 25 is in fluid communication with a source of SCCNC fluid (not shown).
  • [0032]
    Polymer vessel 13 is also in fluid communication with a modifier syringe pump 31 via conduit 33 which intersects with conduit 27 a and 27 b at junction 35. Modifier syringe pump 31 is in communication with a source of modifiers and/or entrainers (not shown).
  • [0033]
    Polymer vessel 13 is loaded with polymer. And, polymer vessel receives SCNCC fluid from SCNCC pump 25 via conduits 27 a and 27 b. Polymer vessel 13 receives modifiers and/or entrainers from Modifier pump 31 via conduit 33. Polymer is dissolved in the SCNCC fluid and modifier to form a polymer solution.
  • [0034]
    Polymer vessel 13 is in fluid communication with admixture chamber 17 via conduits 37 and 39. Admixture chamber 17 is also in fluid communication with bioactive injection assembly 15. Bioactive injection assembly 15 comprises bioactive syringe pump 43, a source of bioactive material (not shown) and conduit 45. Bioactive syringe pump 43 is in communication with a source of bioactive material and pressurizes and compels such material through conduit 45. Conduit 45 is in communication with admixture chamber via conduits 39 which intersects conduit 45 at junction 47. Preferably junction 47 is a mixing “T”.
  • [0035]
    Admixture vessel 17 is in the nature of an inline mixer and thoroughly mixes incoming streams from the polymer vessel 13 and bioactive injection assembly 15. Admixture vessel 17 is in communication with orifice nozzle 21 via conduit 49. Orifice nozzle 21 is in the nature of a back pressure regulator and has a nozzle defining one or more orifices which discharge into depressurization vessel 19 via conduit 51. Preferably orifice nozzle 21 controls pressure and decompression rates.
  • [0036]
    The operating pressure of the system can be preset at a precise level via a computerized controller (not shown) that is part of the syringe pumps. Temperature control in the system is achieved by enclosing the apparatus 11 in ¼″ Lexan sheet while utilizing a Neslab heating/cooling system coupled with a heat exchanger (not shown) to maintain uniform temperature throughout the system.
  • [0037]
    In a typical experimental run, polymeric materials were first packed into the polymer vessel 13. SCCNC and an ethanolic solution of insulin were charged into the SCNCC syringe pumps 25 and 31, respectively, and brought to the desired operating pressure. In the alternative, a ethanol solution of insulin is charged into bioactive syringe pump 43.
  • [0038]
    The system was then pressurized with the SCCNC (supercritical fluid (SCF) and cosolvent) via SCNCC syringe pump 25 to the pressure level equal to that set in modifier syringe pump 31 and bioactive syringe pump 43, and maintained at this level with the nozzle orifice 21. The dynamic operating mode for all pumps were set so that each pump can be operated at its own desired flow rate. The SCCNC stream flowed through the polymer vessel 13, dissolved polymer and contacted the insulin stream at junction 47. The mixture of SCCNC, insulin and polymeric materials was then passed through admixture chamber 17 for further mixing. Finally, the mixed solution entered orifice nozzle 21 and was injected into a 0.1% PVA aqueous solution in the depressurization vessel 19. As a result of supercritical fluid decompression, polymeric spheres containing insulin are formed in the PVA aqueous solution and the expanded supercritical fluid exited the system via a vent line on the depressurization vessel 19.
  • EXAMPLES
  • [0039]
    For preliminary experiments, 50:50 mixtures of poly(D,L-lactic glycolic acid) with molecular weights of 3,000, 50,000 and 100,000 (Sigma Chemicals, St. Louis, Mo.) were utilized. For most microsphere and some solubility experiments, Medisorbn® biodegradable polymers (Alkermes, Inc., Cincinnati, Ohio) were utilized. The specifications on the Medisorb polymers are listed in Table 2. “A” indicates that the polymers contain a free carboxylic acid group on the carboxyl end of the polymer chain; “M” indicates an ester end group.
    TABLE 2
    Specifications of Medisorb ® Biodegradable Polymers
    Medisorb Inherent Viscosity Approx. MW DL-lactide/glycolide
    Polymer (dL/g) (Kd) mole ratio
    5050DL2A 0.15 12.3 53/47
    5050DL2M 0.18 17.3 54/46
    5050DL3A 0.25-0.33 20-28 54/46
  • [0040]
    Other materials utilized include insulin, cytochrome-C, tetanus and diphtheria toxoids, ethyl alcohol (USP grade), distilled water, and polyvinyl alcohol (PVA).
  • Example 1 SCCNC Polymer Microspheres/Nanospheres Formed with Near-Critical Propane
  • [0041]
    Polymer microspheres/nanospheres were formed with 50:50 PLGA obtained from Sigma Chemicals (St. Louis, Mo.) in the SCCNC polymer sphere apparatus running in the continuous mode. The polymer microspheres/nanospheres were formed by injecting the SCCNC polymer solution into distilled water. The resulting product was observed under a light microscope, and the particle sizes were measured in a Coulter 4MD sub-micron particle size analyzer. The volume of distilled water used in PMF-03a was about half of that used in PMF-01 resulting in a more concentrated microsphere solution and a different particle size distribution. Some of these results are presented in Table 3.
    TABLE 3
    SCCNC Polymer Microspheres/Nanospheres Formed with 50:50 PLGA at 40° C.
    Pressure Flow Rate Small Medium Large
    Run No. SCCNC (psig) (ml/min) Size (nm)/% Size (nm)/% Size (nm)/%
    PMF-01  C3H8 2,000 1.0 99 (26%) 336 (74%)
    PMF-02a C3H8 4,000 1.0 120 (34%) 2,120 (66%)
    PMF-02b C3H8 5,000 3.0 33 (57%) 282 (15%) 10,000 (29%) 
    PMF-03a C3H8 2,000 1.0 291 (76%) 1,770 (24%)
    PMF-03b C3H8 2,000 4.0 169 (33%)   852 (67%)
  • Example 2 Protein Antigencity in Polymer Microspheres formed by Different SCCNC
  • [0042]
    Experiments were performed to encapsulate tetanus toxoid (TT) and diphtheria toxoid (DT) vaccine antigens in 50:50 PLGA polymer microspheres formed by SCCNC carbon dioxide and propane. In these tests, DT and TT were each separately treated with supercritical carbon dioxide with 10% (v/v) cosolvent ethanol, and near-critical propane in the presence of PLGA. The pressure and temperature were around 3,000 psig and 30-35° C. respectively. The protein and antigenicity activities were performed by micro BCA assay and a sandwich-type capture ELISA [Gupta, R. K., Siber, G. R., Alonso, M. J. and Langer, R., in Modern Approaches To New Vaccines Including Prevention of AIDS. Ed. by Ginsberg, H. S., Brown, F., Chanock, R. M. and Lerner, R. A. Cold Spring Harbor Laboratory, Press, 1993] assay. The results of this study are summarized in Table 4.
    TABLE 4
    Protein Content and Antigencity of Tetanus Toxoid and Diphtheria
    Toxoid in PLGA Polymer Microspheres formed by Different SCCNC
    Sample
    No. Toxoid SCCNC Protein (mg/ml) ELISA (Lf/ml)
    1 Diphtheria Control 21.07 7.35
    2 Diphtheria CO2/ethanol  1.49 0.13
    3 Diphtheria C3H8 18.63 6.55
    4 Tetanus Control 30.44 9.0 
    5 Tetanus CO2/ethanol  9.81 0.44
  • [0043]
    Both diphtheria and tetanus toxoids lost most of their antigenicity after being treated with SCCNC carbon dioxide/ethanol mixtures. These losses are probably due to the fact that the acid/base equilibrium shifted due to the formation of carbonic acid when the aqueous protein is exposed to carbon dioxide. This shift can drastically reduce pH if the solution is insufficiently buffered. Both diphtheria and tetanus toxoids will denature at pH levels below 5.0. Thus, carbon dioxide may not be the best candidate for a SCCNC solvent for acid pH sensitive proteins. Propane, on the other hand, did little damage to the diphtheria toxoid because it has negligible impact on the acid/base equilibrium of the aqueous protein.
  • Example 3 Protein (Insulin) Encapsulation by SCCNC Polymer Microspheres/Nanospheres
  • [0044]
    Experiments were conducted to encapsulate insulin in polymer microspheres/nanospheres utilizing supercritical carbon dioxide. Insulin, which has an isoelectric point of 3.65, is stable at acid pHs. In these experiments, a feed solution of 0.1 mg/ml insulin in 90% ethanol:10% water was utilized. The supercritical carbon dioxide was pumped at a rate of 1 ml/min, the cosolvent pump at 0.1 ml/min, and the insulin solution at 0.5 ml/min. The resultant mixture was injected into 8 ml of 1% PVA solution for 30 minutes. The results of these experiments are summarized in Table 5.
    TABLE 5
    Polymer Microspheres/Nanospheres Formed with Medisorb
    Polymers and Insulin in SCCNC Carbon Dioxide/Cosolvent
    at 3,000 psig and 50° C.
    Small Medium Large
    Run No. Polymer SCCNC Size (nm) Size (nm) Size (nm)/%
    MS-09 DL2A CO2/ 750 (22%)   5,250 (77%)
    ethanol
    MS-10 DL2M CO2/ 634 (100%)
    ethanol
    MS-11 DL3A CO2/ 300 (49%)  10,000 (51%)
    ethanol
    MS-12 DL2A CO2/ 326 (100%)
    acetone
  • [0045]
    The data in Table 5 indicates that the Medisorb DL2M and DL2A bioadhesive polymers formed relatively uniform particle size distributions in the SCCNC CO2/ethanol and SCCNC CO2/acetone systems, respectively, at 3,000 psig and 50° C.
  • Example 4 Release of Cytochrome-C from SCCNC Polymer Microspheres/Nanospheres
  • [0046]
    In order to establish conditions for the encapsulation of proteins in uniform microspheres and their renlease characteristics, several experiments were conducted to encapsulate cytochrome-C in polymer microspheres/nanospheres utilizing supercritical carbon dioxide. In these experiments, a feed solution of 0.1 mg/ml cytochrome-C in 99% ethanol: 1% water was utilized. The supercritical carbon dioxide was pumped at a rate of 1.0 ml/min, the cosolvent pump at 0.1 ml/min, and the insulin solution at 0.5 ml/min. The resultant mixture was injected into 8 ml of 1% PVA solution for 30 minutes. The results of some of these experiments are summarized in Table 6.
    TABLE 6
    Polymer Microspheres/Nanospheres Formed with Medisorb Polymers and
    Cytochrome-C in SCCNC Carbon Dioxide and Propane at 3,000 psig
    Temp. Small Medium Large
    Run No. Polymer SCCNC (° C.) Size (nm) Size (nm) Size (nm)
    MS-19 DL3A CO2/10% ethanol 45 318 (100%)
    MS-21 DL2A CO2/10% ethanol 45 292 (100%)
    MS-22 DL2A CO2/10% acetone 45 267 (100%)
    MS-23 DL2M CO2/10% ethanol 45 239 (100%)
    MS-24 DL2A C3H8/10% acetone 30 187 (100%)
    MS-25 DL2A C3H8/3% acetone 40 418 (100%)
  • [0047]
    Some of the size distributions were quite narrow while others were broad. Some of the charts indicate the presence of “dust” which are particles that are larger than 10 micron in size. Most of these particles, from microscopic observations, appear to be excess polymer. These large polymer particles were removed by vacuum filtration prior to solvent evaporation and drying to harden the polymer microspheres/nanospheres. In experiments MS-24 and MS-25, the supercritical fluid and cosolvent pumps were kept in operation for 180 minutes after the feed pump was turned off to ensure that all the protein had been displaced from the high pressure circulation loop.
  • [0048]
    The release characteristics of MS-25 were evaluated by suspending the dried microspheres in 4 ml of PBS at a pH of 7.4. Absorption of the solution was then measured at 408 nm and over the 350 to 450 nm range at different time intervals. Concentration was determined from a standard curve. The release characteristics of MS-25 over a 5½ hour period is shown in FIG. 2.
  • Example 5 Release of Insulin from SCCNC Polymer Microspheres/Nanospheres
  • [0049]
    In this example, a feed solution of 0.1 mg/ml insulin in 90% ethanol:10% water was utilized. Supercritical carbon dioxide was pumped at a rate of 1.0 ml/min, the cosolvent pump at 0.1 ml/min, and the insulin solution at 0.5 ml/min. The resultant mixture was injected into 8 ml of 1 % PVA solution for 30 minutes. The supercritical fluid and cosolvent pumps were kept in operation for 180 minutes after the feed pump was turned off to ensure that all the protein had been displaced from the high pressure circulation loop. The release characteristics of insulin in this experiment MS-27 was evaluated by suspending the dried microspheres in 4 ml of PBS at a pH of 7.4. Absorption of the solution was then measured at 280 nm and over the 250 to 350 nm range at different time intervals. The release characteristics of MS-27 over a 5-hour period are shown in FIG. 3.
  • [0050]
    It is intended that the matter contained in the preceding description be interpreted in an illustrative rather than a limiting sense.
Patent Citations
Cited PatentFiling datePublication dateApplicantTitle
US4582731 *Sep 1, 1983Apr 15, 1986Battelle Memorial InstituteSupercritical fluid molecular spray film deposition and powder formation
US4734451 *Mar 12, 1986Mar 29, 1988Battelle Memorial InstituteSupercritical fluid molecular spray thin films and fine powders
US5043280 *Dec 21, 1988Aug 27, 1991Schwarz Pharma AgMethod and apparatus for the manufacture of a product having a substance embedded in a carrier
US5554382 *May 28, 1993Sep 10, 1996Aphios CorporationMethods and apparatus for making liposomes
US5766637 *Oct 8, 1996Jun 16, 1998University Of DelawareMicroencapsulation process using supercritical fluids
US6124226 *Apr 19, 1999Sep 26, 2000Union Carbide Chemicals & Plastics Technology CorporationProcess for forming a catalyst, catalyst support or catalyst precursor with compressed fluids
Referenced by
Citing PatentFiling datePublication dateApplicantTitle
US7176304May 11, 2004Feb 13, 2007Mcswiggen JamesRNA interference mediated inhibition of vascular endothelial growth factor and vascular endothelial growth factor receptor gene expression using short interfering nucleic acid (siNA)
US7517864Dec 9, 2005Apr 14, 2009Sirna Therapeutics, Inc.RNA interference mediated inhibition of vascular endothelial growth factor and vascular endothelial growth factor receptor gene expression using short interfering nucleic acid (siNA)
US7923547Apr 12, 2011Sirna Therapeutics, Inc.RNA interference mediated inhibition of gene expression using chemically modified short interfering nucleic acid (siNA)
US7956176Jun 7, 2011Sirna Therapeutics, Inc.RNA interference mediated inhibition of gene expression using chemically modified short interfering nucleic acid (siNA)
US7989612Aug 2, 2011Sirna Therapeutics, Inc.RNA interference mediated inhibition of gene expression using chemically modified short interfering nucleic acid (siNA)
US8202979Jun 19, 2012Sirna Therapeutics, Inc.RNA interference mediated inhibition of gene expression using chemically modified short interfering nucleic acid
US8273866Nov 24, 2003Sep 25, 2012Merck Sharp & Dohme Corp.RNA interference mediated inhibition of gene expression using chemically modified short interfering nucleic acid (SINA)
US8299236Sep 8, 2008Oct 30, 2012Marina Biotech, Inc.Compositions and methods for enhancing delivery of nucleic acids into cells and for modifying expression of target genes in cells
US8377448Jan 14, 2010Feb 19, 2013The Board Of Trustees Of The Leland Standford Junior UniversityCD47 related compositions and methods for treating immunological diseases and disorders
US8664194May 21, 2013Mar 4, 2014Moderna Therapeutics, Inc.Method for producing a protein of interest in a primate
US8680069May 18, 2013Mar 25, 2014Moderna Therapeutics, Inc.Modified polynucleotides for the production of G-CSF
US8710200Apr 2, 2012Apr 29, 2014Moderna Therapeutics, Inc.Engineered nucleic acids encoding a modified erythropoietin and their expression
US8710209Dec 8, 2010Apr 29, 2014Nitto Denko CorporationModulation of HSP47 expression
US8754062May 21, 2013Jun 17, 2014Moderna Therapeutics, Inc.DLIN-KC2-DMA lipid nanoparticle delivery of modified polynucleotides
US8822663Aug 5, 2011Sep 2, 2014Moderna Therapeutics, Inc.Engineered nucleic acids and methods of use thereof
US8846894Feb 16, 2007Sep 30, 2014Sirna Therapeutics, Inc.RNA interference mediated inhibition of gene expression using chemically modified short interfering nucleic acid (siNA)
US8980864Dec 20, 2013Mar 17, 2015Moderna Therapeutics, Inc.Compositions and methods of altering cholesterol levels
US8999380Mar 9, 2013Apr 7, 2015Moderna Therapeutics, Inc.Modified polynucleotides for the production of biologics and proteins associated with human disease
US9044420Apr 6, 2012Jun 2, 2015Immune Design Corp.Immunogenic compositions and methods of using the compositions for inducing humoral and cellular immune responses
US9050297Dec 16, 2013Jun 9, 2015Moderna Therapeutics, Inc.Modified polynucleotides encoding aryl hydrocarbon receptor nuclear translocator
US9061059Feb 3, 2014Jun 23, 2015Moderna Therapeutics, Inc.Modified polynucleotides for treating protein deficiency
US9089604Feb 3, 2014Jul 28, 2015Moderna Therapeutics, Inc.Modified polynucleotides for treating galactosylceramidase protein deficiency
US9095552Dec 12, 2013Aug 4, 2015Moderna Therapeutics, Inc.Modified polynucleotides encoding copper metabolism (MURR1) domain containing 1
US9107886Dec 12, 2013Aug 18, 2015Moderna Therapeutics, Inc.Modified polynucleotides encoding basic helix-loop-helix family member E41
US9114113Dec 12, 2013Aug 25, 2015Moderna Therapeutics, Inc.Modified polynucleotides encoding citeD4
US9149506Dec 16, 2013Oct 6, 2015Moderna Therapeutics, Inc.Modified polynucleotides encoding septin-4
US9181319May 6, 2014Nov 10, 2015Moderna Therapeutics, Inc.Engineered nucleic acids and methods of use thereof
US9181551Oct 14, 2014Nov 10, 2015Sirna Therapeutics, Inc.RNA interference mediated inhibition of gene expression using chemically modified short interfering nucleic acid (siNA)
US9186372May 21, 2013Nov 17, 2015Moderna Therapeutics, Inc.Split dose administration
US9192651Mar 9, 2013Nov 24, 2015Moderna Therapeutics, Inc.Modified polynucleotides for the production of secreted proteins
US9206424Mar 18, 2014Dec 8, 2015Nitto Denko CorporationModulation of HSP47 expression
US9216205Dec 16, 2013Dec 22, 2015Moderna Therapeutics, Inc.Modified polynucleotides encoding granulysin
US9220755Dec 13, 2013Dec 29, 2015Moderna Therapeutics, Inc.Modified polynucleotides for the production of proteins associated with blood and lymphatic disorders
US9220792Dec 11, 2013Dec 29, 2015Moderna Therapeutics, Inc.Modified polynucleotides encoding aquaporin-5
US9221891Mar 15, 2013Dec 29, 2015Moderna Therapeutics, Inc.In vivo production of proteins
US9226900Jul 10, 2009Jan 5, 2016Critical Pharmaceuticals LimitedProcess for preparing microparticles
US9233141Dec 12, 2013Jan 12, 2016Moderna Therapeutics, Inc.Modified polynucleotides for the production of proteins associated with blood and lymphatic disorders
US9254311Mar 9, 2013Feb 9, 2016Moderna Therapeutics, Inc.Modified polynucleotides for the production of proteins
US9255129Dec 16, 2013Feb 9, 2016Moderna Therapeutics, Inc.Modified polynucleotides encoding SIAH E3 ubiquitin protein ligase 1
US9260471Oct 25, 2011Feb 16, 2016Sirna Therapeutics, Inc.RNA interference mediated inhibition of gene expression using short interfering nucleic acids (siNA)
US9271996May 18, 2013Mar 1, 2016Moderna Therapeutics, Inc.Formulation and delivery of PLGA microspheres
US9283287Apr 23, 2015Mar 15, 2016Moderna Therapeutics, Inc.Modified polynucleotides for the production of nuclear proteins
US9295689May 18, 2013Mar 29, 2016Moderna Therapeutics, Inc.Formulation and delivery of PLGA microspheres
US9301993Dec 16, 2013Apr 5, 2016Moderna Therapeutics, Inc.Modified polynucleotides encoding apoptosis inducing factor 1
US9303079Mar 9, 2013Apr 5, 2016Moderna Therapeutics, Inc.Modified polynucleotides for the production of cytoplasmic and cytoskeletal proteins
US9334328Jan 11, 2013May 10, 2016Moderna Therapeutics, Inc.Modified nucleosides, nucleotides, and nucleic acids, and uses thereof
US20040192626 *May 23, 2003Sep 30, 2004Mcswiggen JamesRNA interference mediated inhibition of gene expression using chemically modified short interfering nucleic acid (siNA)
US20040198682 *Oct 10, 2003Oct 7, 2004Mcswiggen JamesRNA interference mediated inhibition of placental growth factor gene expression using short interfering nucleic acid (siNA)
US20040209831 *Sep 16, 2003Oct 21, 2004Mcswiggen JamesRNA interference mediated inhibition of hepatitis C virus (HCV) gene expression using short interfering nucleic acid (siNA)
US20040231231 *Apr 1, 2004Nov 25, 2004Cataldo Dominic A.Use of colloidal clays for sustained release of active ingredients
US20050032733 *Apr 16, 2004Feb 10, 2005Sirna Therapeutics, Inc.RNA interference mediated inhibition of gene expression using chemically modified short interfering nucleic acid (SiNA)
US20050054596 *Jan 26, 2004Mar 10, 2005Mcswiggen JamesRNA interference mediated inhibition of vascular endothelial growth factor and vascular endothelial growth factor receptor gene expression using short interfering nucleic acid (siNA)
US20050054598 *Apr 23, 2004Mar 10, 2005Sirna Therapeutics, Inc.RNA interference mediated inhibition hairless (HR) gene expression using short interfering nucleic acid (siNA)
US20050079610 *Aug 20, 2004Apr 14, 2005Sirna Therapeutics, Inc.RNA interference mediated inhibition of Fos gene expression using short interfering nucleic acid (siNA)
US20050084533 *Mar 10, 2003Apr 21, 2005Howdle Steven M.Polymer composite with internally distributed deposition matter
US20050096284 *Feb 20, 2004May 5, 2005Sirna Therapeutics, Inc.RNA interference mediated treatment of polyglutamine (polyQ) repeat expansion diseases using short interfering nucleic acid (siNA)
US20050119211 *Jun 16, 2004Jun 2, 2005Sirna Therapeutics, Inc.RNA mediated inhibition connexin gene expression using short interfering nucleic acid (siNA)
US20050119212 *Jun 18, 2004Jun 2, 2005Sirna Therapeutics, Inc.RNA interference mediated inhibition of FAS and FASL gene expression using short interfering nucleic acid (siNA)
US20050124566 *Jun 28, 2004Jun 9, 2005Sirna Therapeutics, Inc.RNA interference mediated inhibition of myostatin gene expression using short interfering nucleic acid (siNA)
US20050124567 *Jul 1, 2004Jun 9, 2005Sirna Therapeutics, Inc.RNA interference mediated inhibition of TRPM7 gene expression using short interfering nucleic acid (siNA)
US20050124568 *Jul 9, 2004Jun 9, 2005Sirna Therapeutics, Inc.RNA interference mediated inhibition of acetyl-CoA-carboxylase gene expression using short interfering nucleic acid (siNA)
US20050136436 *Aug 19, 2004Jun 23, 2005Sirna Therapeutics, Inc.RNA interference mediated inhibition of G72 and D-amino acid oxidase (DAAO) gene expression using short interfering nucleic acid (siNA)
US20050137153 *May 6, 2004Jun 23, 2005Sirna Therapeutics, Inc.RNA interference mediated inhibition of alpha-1 antitrypsin (AAT) gene expression using short interfering nucleic acid (siNA)
US20050137155 *Jun 3, 2004Jun 23, 2005Sirna Therapeutics, Inc.RNA interference mediated treatment of Parkinson disease using short interfering nucleic acid (siNA)
US20050143333 *Jun 9, 2004Jun 30, 2005Sirna Therapeutics, Inc.RNA interference mediated inhibition of interleukin and interleukin receptor gene expression using short interfering nucleic acid (SINA)
US20050153914 *Aug 16, 2004Jul 14, 2005Sirna Therapeutics, Inc.RNA interference mediated inhibition of MDR P-glycoprotein gene expression using short interfering nucleic acid (siNA)
US20050153915 *Aug 19, 2004Jul 14, 2005Sirna Therapeutics, Inc.RNA interference mediated inhibition of early growth response gene expression using short interfering nucleic acid (siNA)
US20050159376 *May 12, 2004Jul 21, 2005Slrna Therapeutics, Inc.RNA interference mediated inhibition 5-alpha reductase and androgen receptor gene expression using short interfering nucleic acid (siNA)
US20050159380 *Aug 19, 2004Jul 21, 2005Sirna Therapeutics, Inc.RNA interference mediated inhibition of angiopoietin gene expression using short interfering nucleic acid (siNA)
US20050159381 *Aug 20, 2004Jul 21, 2005Sirna Therapeutics, Inc.RNA interference mediated inhibition of chromosome translocation gene expression using short interfering nucleic acid (siNA)
US20050159382 *Aug 19, 2004Jul 21, 2005Sirna Therapeutics, Inc.RNA interference mediated inhibition of polycomb group protein EZH2 gene expression using short interfering nucleic acid (siNA)
US20050164224 *Jul 16, 2004Jul 28, 2005Sirna Therapeutics, Inc.RNA interference mediated inhibition of cyclin D1 gene expression using short interfering nucleic acid (siNA)
US20050164966 *Aug 16, 2004Jul 28, 2005Sirna Therapeutics, Inc.RNA interference mediated inhibition of type 1 insulin-like growth factor receptor gene expression using short interfering nucleic acid (siNA)
US20050164967 *Aug 19, 2004Jul 28, 2005Sirna Therapeutics, Inc.RNA interference mediated inhibition of platelet-derived endothelial cell growth factor (ECGF1) gene expression using short interfering nucleic acid (siNA)
US20050164968 *Aug 20, 2004Jul 28, 2005Sirna Therapeutics, Inc.RNA interference mediated inhibition of ADAM33 gene expression using short interfering nucleic acid (siNA)
US20050171039 *May 11, 2004Aug 4, 2005Sirna Therapeutics, Inc.RNA interference mediated inhibition of vascular endothelial growth factor and vascular endothelial growth factor receptor gene expression using short interfering nucleic acid (siNA)
US20050176664 *Aug 17, 2004Aug 11, 2005Sirna Therapeutics, Inc.RNA interference mediated inhibition of cholinergic muscarinic receptor (CHRM3) gene expression using short interfering nucleic acid (siNA)
US20050176666 *Aug 20, 2004Aug 11, 2005Sirna Therapeutics, Inc.RNA interference mediated inhibition of GPRA and AAA1 gene expression using short interfering nucleic acid (siNA)
US20050182006 *Jul 30, 2004Aug 18, 2005Sirna Therapeutics, IncRNA interference mediated inhibition of protein kinase C alpha (PKC-alpha) gene expression using short interfering nucleic acid (siNA)
US20050182007 *Aug 20, 2004Aug 18, 2005Sirna Therapeutics, Inc.RNA interference mediated inhibition of interleukin and interleukin receptor gene expression using short interfering nucleic acid (SINA)
US20050182009 *Aug 20, 2004Aug 18, 2005Sirna Therapeutics, Inc.RNA interference mediated inhibition of NF-Kappa B / REL-A gene expression using short interfering nucleic acid (siNA)
US20050187174 *Aug 20, 2004Aug 25, 2005Sirna Therapeutics, Inc.RNA interference mediated inhibition of intercellular adhesion molecule (ICAM) gene expression using short interfering nucleic acid (siNA)
US20050191638 *Apr 14, 2004Sep 1, 2005Sirna Therapeutics, Inc.RNA interference mediated treatment of polyglutamine (polyQ) repeat expansion diseases using short interfering nucleic acid (siNA)
US20050196765 *Jul 23, 2004Sep 8, 2005Sirna Therapeutics, Inc.RNA interference mediated inhibition of checkpoint Kinase-1 (CHK-1) gene expression using short interfering nucleic acid (siNA)
US20050196767 *Aug 20, 2004Sep 8, 2005Sirna Therapeutics, Inc.RNA interference mediated inhibition of GRB2 associated binding protein (GAB2) gene expression using short interfering nucleic acis (siNA)
US20050203040 *Aug 16, 2004Sep 15, 2005Sirna Therapeutics, Inc.RNA interference mediated inhibition of vascular cell adhesion molecule (VCAM) gene expression using short interfering nucleic acid (siNA)
US20050209179 *Jun 25, 2004Sep 22, 2005Sirna Therapeutics, Inc.RNA interference mediated treatment of Alzheimer's disease using short interfering nucleic acid (siNA)
US20050209180 *Sep 15, 2004Sep 22, 2005Sirna Therapeutics, Inc.RNA interference mediated inhibition of hepatitis C virus (HCV) expression using short interfering nucleic acid (siNA)
US20050222064 *Jul 9, 2004Oct 6, 2005Sirna Therapeutics, Inc.Polycationic compositions for cellular delivery of polynucleotides
US20050222066 *Oct 12, 2004Oct 6, 2005Sirna Therapeutics, Inc.RNA interference mediated inhibition of vascular endothelial growth factor and vascular endothelial growth factor receptor gene expression using short interfering nucleic acid (siNA)
US20050227935 *Aug 20, 2004Oct 13, 2005Sirna Therapeutics, Inc.RNA interference mediated inhibition of TNF and TNF receptor gene expression using short interfering nucleic acid (siNA)
US20050233329 *Dec 3, 2003Oct 20, 2005Sirna Therapeutics, Inc.Inhibition of gene expression using duplex forming oligonucleotides
US20050233344 *Aug 20, 2004Oct 20, 2005Sirna Therapeutics, Inc.RNA interference mediated inhibition of platelet derived growth factor (PDGF) and platelet derived growth factor receptor (PDGFR) gene expression using short interfering nucleic acid (siNA)
US20050233996 *Apr 26, 2004Oct 20, 2005Sirna Therapeutics, Inc.RNA interference mediated inhibition of hairless (HR) gene expression using short interfering nucleic acid (siNA)
US20050233997 *Aug 17, 2004Oct 20, 2005Sirna Therapeutics, Inc.RNA interference mediated inhibition of matrix metalloproteinase 13 (MMP13) gene expression using short interfering nucleic acid (siNA)
US20050256068 *Aug 20, 2004Nov 17, 2005Sirna Therapeutics, Inc.RNA interference mediated inhibition of stearoyl-CoA desaturase (SCD) gene expression using short interfering nucleic acid (siNA)
US20050267058 *Aug 20, 2004Dec 1, 2005Sirna Therapeutics, Inc.RNA interference mediated inhibition of placental growth factor gene expression using short interfering nucleic acid (sINA)
US20050282188 *Apr 4, 2005Dec 22, 2005Sirna Therapeutics, Inc.RNA interference mediated inhibition of gene expression using short interfering nucleic acid (siNA)
US20050287128 *Feb 9, 2005Dec 29, 2005Sirna Therapeutics, Inc.RNA interference mediated inhibition of TGF-beta and TGF-beta receptor gene expression using short interfering nucleic acid (siNA)
US20050288242 *Aug 20, 2004Dec 29, 2005Sirna Therapeutics, Inc.RNA interference mediated inhibition of RAS gene expression using short interfering nucleic acid (siNA)
US20060019913 *Jan 6, 2005Jan 26, 2006Sirna Therapeutics, Inc.RNA interference mediated inhibtion of protein tyrosine phosphatase-1B (PTP-1B) gene expression using short interfering nucleic acid (siNA)
US20060025361 *Jan 14, 2005Feb 2, 2006Sirna Therapeutics, Inc.RNA interference mediated inhibition of protein tyrosine phosphatase-1B (PTP-1B) gene expression using short interfering nucleic acid (siNA)
US20060142226 *Aug 19, 2004Jun 29, 2006Sirna Therapeutics, Inc.RNA interference mediated inhibition of cholesteryl ester transfer protein (CETP) gene expression using short interfering nucleic acid (siNA)
US20060211642 *Dec 19, 2005Sep 21, 2006Sirna Therapeutics, Inc.RNA inteference mediated inhibition of hepatitis C virus (HVC) gene expression using short interfering nucleic acid (siNA)
US20060216747 *Jun 6, 2006Sep 28, 2006Sirna Therapeutics, Inc.RNA interference mediated inhibition of checkpoint kinase-1 (CHK-1) gene expression using short interfering nucleic acid (siNA)
US20060217332 *Dec 9, 2005Sep 28, 2006Sirna Therapeutics, Inc.RNA interference mediated inhibition of vascular endothelial growth factor and vascular endothelial growth factor receptor gene expression using short interfering nucleic acid (siNA)
US20060217334 *Feb 21, 2006Sep 28, 2006Sirna Therapeutics, Inc.RNA interference mediated inhibition of gene expression using chemically modified short interfering nucleic acid (siNA)
US20060217335 *Feb 21, 2006Sep 28, 2006Sirna Therapeutics, Inc.RNA interference mediated inhibition of gene expression using chemically modified short interfering nucleic acid (siNA)
US20060217336 *Feb 21, 2006Sep 28, 2006Sirna Therapeutics, Inc.RNA interference mediated inhibition of gene expression using chemically modified short interfering nucleic acid (siNA)
US20060217337 *Feb 21, 2006Sep 28, 2006Sirna Therapeutics, Inc.RNA interference mediated inhibition of gene expression using chemically modified short interfering nucleic acid (siNA)
US20060241075 *Oct 26, 2005Oct 26, 2006Sirna Therapeutics, Inc.RNA interference mediated inhibition of desmoglein gene expression using short interfering nucleic acid (siNA)
US20060247428 *Feb 21, 2006Nov 2, 2006Sirna Therapeutics, Inc.RNA interference mediated inhibition of gene expression using chemically modified short interfering nucleic acid (siNA)
US20060275903 *Aug 11, 2006Dec 7, 2006Sirna Therapeutics, Inc.RNA interference mediated inhibition of gene expression using chemically modified short interfering nucleic acid (siNA)
US20060281175 *Aug 11, 2006Dec 14, 2006Sirna Therapeutics, Inc.RNA interference mediated inhibition of gene expression using chemically modified short interfering nucleic acid (siNA)
US20060287266 *Feb 21, 2006Dec 21, 2006Sirna Therapeutics, Inc.RNA interference mediated ihibition of gene expression using chemically modified short interfering nucleic acid (siNA)
US20060292691 *Aug 11, 2006Dec 28, 2006Sirna Therapeutics, Inc.RNA interference mediated inhibition of gene expression using chemically modified short interfering nucleic acid (siNA)
US20060293272 *Aug 4, 2006Dec 28, 2006Sirna Therapeutics, Inc.RNA interference mediated inhibition of gene expression using chemically modified short interfering nucleic acid (siNA)
US20070004667 *Aug 11, 2006Jan 4, 2007Sirna Therapeutics, Inc.RNA interference mediated inhibition of gene expression using chemically modified short interfering nucleic acid (siNA)
US20070102836 *Jan 20, 2005May 10, 2007Japan Science And Technology AgencyMethod for producing fine particles using method of rapid expansion into poor solvent from supercritical fluid
US20070160980 *Mar 6, 2006Jul 12, 2007Sirna Therapeutics, Inc.RNA interference mediated inhibition of gene expression using short interfering nucleic acid (siNA)
US20070167393 *Feb 16, 2007Jul 19, 2007Sirna Therapeutics, Inc.RNA INTERFERENCE MEDIATED INHIBITION OF GENE EXPRESSION USING CHEMICALLY MODIFIED SHORT INTERFERING NUCLEIC ACID (siNA)
US20070203333 *Sep 18, 2003Aug 30, 2007Mcswiggen JamesRNA interference mediated inhibition of vascular endothelial growth factor and vascular endothelial growth factor receptor gene expression using short interfering nucleic acid (siNA)
US20070270579 *Sep 23, 2005Nov 22, 2007Sirna Therapeutics, Inc.RNA interference mediated inhibition of gene expression using short interfering nucleic acid (siNA)
US20070275923 *Feb 16, 2007Nov 29, 2007Nastech Pharmaceutical Company Inc.CATIONIC PEPTIDES FOR siRNA INTRACELLULAR DELIVERY
US20070276134 *Feb 16, 2007Nov 29, 2007Nastech Pharmaceutical Company Inc.Compositions and methods for complexes of nucleic acids and organic cations
US20070281900 *May 2, 2007Dec 6, 2007Nastech Pharmaceutical Company Inc.COMPOSITIONS AND METHODS FOR LIPID AND POLYPEPTIDE BASED siRNA INTRACELLULAR DELIVERY
US20070293657 *Feb 2, 2007Dec 20, 2007Nastech Pharmaceutical Company Inc.Complexes and methods of forming complexes of ribonucleic acids and peptides
US20080039414 *Oct 23, 2003Feb 14, 2008Sima Therapeutics, Inc.RNA interference mediated inhibition of gene expression using chemically modified short interfering nucleic acid (siNA)
US20080131431 *May 15, 2007Jun 5, 2008Viral Logic Systems Technology Corp.CD47 related compositions and methods for treating immunological diseases and disorders
US20080188430 *Aug 20, 2004Aug 7, 2008Sirna Therapeutics, Inc.RNA interference mediated inhibition of hypoxia inducible factor 1 (HIF1) gene expression using short interfering nucleic acid (siNA)
US20080261304 *Jan 11, 2008Oct 23, 2008Nastech Pharmaceutical Company Inc.Methods and compositions for enhancing delivery of double-stranded rna or a double-stranded hybrid nucleic acid to regulate gene expression in mammalian cells
US20080274275 *Nov 21, 2005Nov 6, 2008Japan Science And Technology AgencyMethod For Preparing Composite Fine Particles
US20090042298 *Sep 8, 2008Feb 12, 2009Mdrna, Inc.Compositions and methods for enhancing delivery of nucleic acids into cells and for modifying expression of target genes in cells
US20090137500 *Nov 24, 2003May 28, 2009Sirna Therapeutics, Inc.RNA interference mediated inhibition of gene expression using chemically modified short interfering nucleic acid (siNA)
US20090176725 *Aug 17, 2006Jul 9, 2009Sirna Therapeutics Inc.Chemically modified short interfering nucleic acid molecules that mediate rna interference
US20100239579 *Jan 14, 2010Sep 23, 2010Viral Logic Systems Technology Corp.CD47 Related Compositions and Methods for Treating Immunological Diseases and Disorders
US20110172141 *Jul 10, 2009Jul 14, 2011Critical Pharmaceuticals LimitedProcess for preparing microparticles
US20110178157 *Dec 8, 2010Jul 21, 2011Nitto Denko Technical CorporationModulation of hsp47 expression
CN104114572A *Dec 14, 2012Oct 22, 2014现代治疗公司Modified nucleoside, nucleotide, and nucleic acid compositions
EP2494993A2May 2, 2008Sep 5, 2012Marina Biotech, Inc.Amino acid lipids and uses thereof
EP2518509A2Mar 5, 2009Oct 31, 2012The Regents of the University of CaliforniaMolecular prognosis and classification of malignant melanoma based upon markers selected from the list consisting of RGS1, NCOA3, SPP1, PHIP.
EP2522752A1Aug 11, 2008Nov 14, 2012Baxter International Inc.IVIG modulation of chemokines for treatment of Multiple Sclerosis, Alzheimer's disease, and Parkinson's disease
EP2522753A1Aug 11, 2008Nov 14, 2012Baxter International Inc.IVIG modulation of chemokines for treatment of Multiple Sclerosis, Alzheimer's disease, and Parkinson's disease
EP2522754A1Aug 11, 2008Nov 14, 2012Baxter International Inc.IVIG modulation of chemokines for treatment of Multiple Sclerosis, Alzheimer's disease, and Parkinson's disease
EP2522755A1Aug 11, 2008Nov 14, 2012Baxter International IncIVIG modulation of chemokines for treatment of Multiple Sclerosis, Alzheimer's disease, and Parkinson's disease
EP2546264A1Jul 15, 2011Jan 16, 2013Fundacio Institut D'Investigacio Biomedica De Bellvitge (Idibell)Compositions and methods for immunomodulation
EP2557089A2Jul 15, 2011Feb 13, 2013Fundació Institut d'Investigació Biomèdica de Bellvitge (IDIBELL)Compositions and methods for immunomodulation
EP2589961A2Sep 6, 2007May 8, 2013The Regents of the University of CaliforniaMolecular diagnosis and classification of malignant melanoma
EP2617434A1Jan 20, 2012Jul 24, 2013Laboratorios Del. Dr. Esteve, S.A.HIV-1 integrase deficient immunogens and methods for loading dendritic cells with said immunogens
EP2679998A1Sep 6, 2007Jan 1, 2014The Regents of the University of CaliforniaMolecular diagnosis and classification of malignant melanoma
EP2679999A1Sep 6, 2007Jan 1, 2014The Regents of the University of CaliforniaMolecular diagnosis and classification of malignant melanoma
EP2680000A1Sep 6, 2007Jan 1, 2014The Regents of the University of CaliforniaMolecular diagnosis and classification of malignant melanoma
EP2902013A1Oct 16, 2009Aug 5, 2015Marina Biotech, Inc.Processes and Compositions for Liposomal and Efficient Delivery of Gene Silencing Therapeutics
EP2924435A2Oct 22, 2008Sep 30, 2015The Regents of The University of CaliforniaBiomarkers for prenatal diagnosis of congenital cytomegalovirus
EP3012324A2Dec 8, 2010Apr 27, 2016Nitto Denko CorporationModulation of hsp47 expression
WO2008137758A2May 2, 2008Nov 13, 2008Mdrna, Inc.Amino acid lipids and uses thereof
WO2009055487A1Oct 22, 2008Apr 30, 2009The Regents Of The University Of CaliforniaBiomarkers for prenatal diagnosis of congenital cytomegalovirus
WO2010107952A2Mar 17, 2010Sep 23, 2010Merck Sharp & Dohme Corp.RNA INTERFERENCE MEDIATED INHIBITION OF CONNECTIVE TISSUE GROWTH FACTOR (CTGF) GENE EXPRESSION USING SHORT INTERFERING NUCLEIC ACID (siNA)
WO2010107955A2Mar 17, 2010Sep 23, 2010Merck Sharp & Dohme Corp.RNA INTERFERENCE MEDIATED INHIBITION OF BTB AND CNC HOMOLOGY 1, BASIC LEUCINE ZIPPER TRANSCRIPTION FACTOR 1 (BACH 1) GENE EXPRESSION USING SHORT INTERFERING NUCLEIC ACID (siNA) SEQUENCE LISTING
WO2010107957A2Mar 17, 2010Sep 23, 2010Merck Sharp & Dohme Corp.RNA INTERFERENCE MEDIATED INHIBITION OF GATA BINDING PROTEIN 3 (GATA3) GENE EXPRESSION USING SHORT INTERFERING NUCLEIC ACID (siNA)
WO2010107958A1Mar 17, 2010Sep 23, 2010Merck Sharp & Dohme Corp.RNA INTERFERENCE MEDIATED INHIBITION OF SIGNAL TRANSDUCER AND ACTIVATOR OF TRANSCRIPTION 6 (STAT6) GENE EXPRESSION USING SHORT INTERFERING NUCLEIC ACID (siNA)
WO2010111464A1Mar 25, 2010Sep 30, 2010Merck Sharp & Dohme Corp.RNA INTERFERENCE MEDIATED INHIBITION OF APOPTOSIS SIGNAL-REGULATING KINASE 1 (ASK1) GENE EXPRESSION USING SHORT INTERFERING NUCLEIC ACID (siNA)
WO2010111468A2Mar 25, 2010Sep 30, 2010Merck Sharp & Dohme Corp.RNA INTERFERENCE MEDIATED INHIBITION OF THE NERVE GROWTH FACTOR BETA CHAIN (NGFß) GENE EXPRESSION USING SHORT INTERFERING NUCLEIC ACID (SINA)
WO2010111471A2Mar 25, 2010Sep 30, 2010Merck Sharp & Dohme Corp.RNA INTERFERENCE MEDIATED INHIBITION OF SIGNAL TRANSDUCER AND ACTIVATOR OF TRANSCRIPTION 1 (STAT1) GENE EXPRESSION USING SHORT INTERFERING NUCLEIC ACID (siNA)
WO2010111490A2Mar 25, 2010Sep 30, 2010Merck Sharp & Dohme Corp.RNA INTERFERENCE MEDIATED INHIBITION OF THE THYMIC STROMAL LYMPHOPOIETIN (TSLP) GENE EXPRESSION USING SHORT INTERFERING NUCLEIC ACID (siNA)
WO2010111497A2Mar 25, 2010Sep 30, 2010Merck Sharp & Dohme Corp.RNA INTERFERENCE MEDIATED INHIBITION OF THE INTERCELLULAR ADHESION MOLECULE 1 (ICAM-1)GENE EXPRESSION USING SHORT INTERFERING NUCLEIC ACID (siNA)
WO2011094759A2Feb 1, 2011Aug 4, 2011The Regents Of The University Of CaliforniaNovel diagnostic and therapeutic targets associated with or regulated by n-cadherin expression and/or epithelial to mesenchymal transition (emt) in prostate cancer and other malignancies
WO2011163436A1Jun 23, 2011Dec 29, 2011Quark Pharmaceuticals, Inc.Double stranded rna compounds to rhoa and use thereof
WO2012044979A2Sep 30, 2011Apr 5, 2012The Goverment Of The United States Of America As Represented By The Secretary Of The Department Of Health And Human ServicesManipulation of stem cell function by p53 isoforms
WO2012047631A2Sep 27, 2011Apr 12, 2012The Children's Hospital Of PhiladelphiaCompositions and methods useful for the treatment and diagnosis of inflammatory bowel disease
WO2012101639A3 *Jan 24, 2012Dec 13, 2012Yissum Research Development Company Of The Hebrew University Of Jerusalem Ltd.Nanoparticles based for dermal and systemic delivery of drugs
WO2012112691A1Feb 15, 2012Aug 23, 2012Immune Design Corp.Methods for enhancing immunogen specific immune responses by vectored vaccines
WO2012118910A2Mar 1, 2012Sep 7, 2012Quark Pharmaceuticals, Inc.Compositions and methods for treating lung disease and injury
WO2012141984A1Apr 6, 2012Oct 18, 2012Immune Design Corp.Immunogenic compositions and methods of using the compositions for inducing humoral and cellular immune responses
WO2013010998A2Jul 16, 2012Jan 24, 2013Fundació Institut D'investigació Biomèdica De Bellvitge (Idibell)Compositions and methods for immunomodulation
WO2013090648A1 *Dec 14, 2012Jun 20, 2013modeRNA TherapeuticsModified nucleoside, nucleotide, and nucleic acid compositions
WO2013106494A1Jan 10, 2013Jul 18, 2013Quark Pharmaceuticals, Inc.Combination therapy for treating hearing and balance disorders
WO2014043289A2Sep 12, 2013Mar 20, 2014Quark Pharmaceuticals, Inc.Double-stranded oligonucleotide molecules to ddit4 and methods of use thereof
WO2014043292A1Sep 12, 2013Mar 20, 2014Quark Pharmaceuticals, Inc.Double-stranded oligonucleotide molecules to p53 and methods of use thereof
WO2014138687A1Mar 7, 2014Sep 12, 2014Irm LlcPeptides and compositions for treatment of joint damage
WO2015020960A1Aug 4, 2014Feb 12, 2015Novartis AgNovel lncrna polynucleotides
WO2015123496A1Feb 13, 2015Aug 20, 2015Immune Design Corp.Immunotherapy of cancer through combination of local and systemic immune stimulation
WO2015175487A1May 12, 2015Nov 19, 2015Novartis AgCompounds and compositions for inducing chondrogenesis
WO2016011083A1Jul 14, 2015Jan 21, 2016Immune Design Corp.Prime-boost regimens with a tlr4 agonist adjuvant and a lentiviral vector
Classifications
U.S. Classification264/14, 264/101, 425/5, 425/6
International ClassificationA61K9/51, A61K9/16, B29B9/12
Cooperative ClassificationB29B2009/125, A61K9/5153, A61K9/5192, B29B9/12, A61K9/1694
European ClassificationA61K9/16P4, B29B9/12, A61K9/51H6D4, A61K9/51P