The present invention relates to medical applications of novel microspheres. Specifically, methods for using novel swellable, deformable microspheres for forming occlusions, bulking tissue, filling voids, delivering medications and absorbing body fluids are provided.
There are a variety of medical procedures that would benefit from the availability of new materials that can be introduced into the body of a mammal for purposes such as tissue bulking, filling voids, forming occlusions, absorbing fluids, and delivering medications. Tissue bulking includes introduction of materials in a collapsed area to provide a filling function, for example in cases where the natural tissue has degenerated such that control of a normal function has deteriorated. Esophageal sphincter and urinary tract control are examples. Void filling involves introduction of materials into an empty space, such as one created by removal of a tissue mass. Void filling may also be used when a tissue cavity is malfunctioning. Occlusions may be imposed to block ducts as well as vasculature. Absorbing fluids and delivering medications is useful in wound treatment, including those caused surgically and accidentally, and involves introduction of materials to stop bleeding, provide padding, deliver medication, and absorb fluids. Such materials are useful especially in emergency situations including accidents and military operations.
Hydrogel microspheres have been used in some of these medical applications. Characteristics of the medical treatment are generally related to the properties of the specific type of hydrogel microspheres used in the treatment. The properties of the hydrogel microspheres are in turn generally dependent upon the materials used in microsphere preparation and the process by which the microspheres were prepared.
U.S. Pat. No. 6,218,440 discloses a process for preparing hydrogel microspheres which produces microspheres that have many cavities joined by interconnecting pores, with cavities at the interior of the material in communication with the surface. In the process disclosed therein, an emulsion is first prepared, and then this emulsion is suspended in an oil medium. The resulting microspheres have high absorption capacity and are said to be useful for adsorption of fluids or chemicals.
U.S. Pat. No. 4,446,261 discloses a process for making hydrogel microspheres which includes dispersing a solution containing monomer, crosslinking agent and initiator in a dispersion medium consisting of hydrocarbons having from 6 to 10 carbon atoms or halogenated aromatic hydrocarbons, with a protective colloid dissolved in this oil material.
U.S. Pat. No. 6,436,424 discloses microspheres suitable for dermal augmentation that are said to swell upon contacting physiological fluids at the injection site to up to four times the average diameter of the microspheres prior to injection. These hydrogel microspheres are said to be made by standard methods of polymerization and microsphere preparation described in the art. The microspheres described in U.S. Pat. No. 6,790,456 are the same as those described in U.S. Pat. No. 6,436,424.
WO 2001072281 discloses hydrogel microspheres as described in U.S. Pat. No. 6,436,424 and U.S. Pat. No. 6,790,456 that are associated with bioactive therapeutic factors such as drugs, genes and diagnostic agents, for use in embolization.
JP1994056676A discloses a suspension used for embolization containing lipidic contrast agent and highly water absorbant hydrogel particles of vinyl alcohol and sodium acrylate polymer that are approximately 1.0 mm in diameter or less.
There remains a need for methods of medical treatment where microspheres with outstanding properties, including high swelling and deformability, are administered to provide effectual tissue bulking, void filling, occlusion forming, fluid absorbing, and medication delivering treatments.
The present invention provides a method for non-vascular occlusion in a mammal comprising administering into a non-vasculature passageway microspheres prepared by a process comprising the following steps:
In another embodiment, the present invention provides a method for tissue bulking in a mammal comprising administering into a thinned or degenerated tissue microspheres prepared by the same process.
In a third embodiment, the present invention provides a method for void filling in a mammal comprising administering in an intra-organ or extra-organ void microspheres prepared by the same process.
In a fourth embodiment, the present invention provides a method for bodily fluid absorption in a mammal comprising administering to a wound, whether accidentally or surgically created, microspheres prepared by the same process.
In a fifth embodiment, the present invention provides a method for medication delivery in a mammal comprising administering microspheres containing a medication prepared by the same process with an additional step of imbibing the microspheres with a medication.
The present invention provides methods of medical treatment using novel high swelling, deformable microspheres. The microspheres have properties of general consistency in size and shape, high density, low fracture, high swell capacity, rapid swell, and deformability following swell. The microspheres are administered to a mammal to provide treatments including tissue bulking, filling voids, forming occlusions, absorbing fluids, and delivering medications. These treatments may be used for a wide range of medical indications. The microspheres are made using a process which is simple, consistent, and produces microspheres with these properties at a high yield. The process for microsphere preparation makes use of a water soluble, low temperature-active azo initiator in an aqueous solution of monomer, crosslinking agent, and emulsifier. A chlorinated organic medium is used in forming a suspension with the aqueous solution. The aqueous solution and organic medium both additionally include protecting colloids. The aqueous solution and organic medium, as well as the mixture of the two, are initially held below the initiation temperature of the azo initiator. The organic medium, which may comprise a chloroform and methylene chloride mixture, has a high enough boiling temperature that the aqueous soluble azo initiator can be activated to cause polymerization producing microspheres.
When an amount, concentration, or other value or parameter is recited herein as either a range, preferred range or a list of upper preferable values and lower preferable values, the recited amount, concentration, or other value or parameter is intended to include all ranges formed from any pair of any upper range limit or preferred value and any lower range limit or preferred value, regardless of whether such ranges are separately disclosed. Where a range of numerical values is recited herein, unless otherwise stated, the range is intended to include the endpoints thereof, and all integers and fractions within the range. It is not intended that the scope of the invention be limited to the specific values recited when defining a range.
The following definitions and abbreviations are to be used for the interpretation of the claims and the specification.
The term “microspheres” or “microsphere” refers to either a population of micron size particles, or an individual particle, depending upon the context in which the word is used, which has a high sphericity measurement. The sphericity measurement of a population of microspheres may be in the range of about 80% to about 100%, with 95% being typical. The microspheres are substantially spherical, although a microsphere population may include some individual particles that have a lower sphericity measurement.
The term “miscible” refers to mixing of two liquids without separation of two phases. In addition, a solid is miscible if a solution made with the solid is miscible with another liquid. Specifically, a liquid monomer may itself be miscible with water. A solid monomer is water miscible when an aqueous solution prepared with the solid monomer can be mixed with water without having a separation of two phases.
The term “substantially chlorinated hydrocarbon” refers to a hydrocarbon that is from 50% to fully chlorinated. Carbon tetrachloride is an example of such a hydrocarbon.
The term “slurry” refers to a composition that is a particulate material in a liquid.
The terms “first suspension” and “second suspension” refer to suspensions formed during the process of preparing microspheres that is described herein.
The term “microsphere suspension” refers to a suspension that contains microspheres which may be administered in a mammal for medical treatment, typically using a catheter and/or needle, for example, in a tissue, cavity, or duct.
The term “deformable” refers to the property of being able to change shape in response to an external pressure. Microspheres are deformable if they do not retain their shape when they are swelled, following uptake of an aqueous medium, and are subjected to pressure.
The term “substantially nonexpendable tubing” refers to tubing that has no visually observable expansion under the testing conditions in which it is used.
The term “swell-control medium” refers to a medium that controls the swell of microspheres, prepared by the present process, such that there is little or no swell. A small amount of swell may occur. However, full swell that is about 50× or more than the original volume does not occur.
The term “anatomical site” refers to any external or internal part of the body of humans or animals.
The term “tissue” refers to any tissue, both living and dead, in humans or animals.
The term “thinned or degenerated tissue” refers to a tissue that has reduced bulk as compared to normal tissue at the same anatomical site. The tissue may have undesirable features such as sagging, wrinkling, or reduced tone.
Microspheres made with the following materials using the process disclosed herein have properties that make them particularly useful in medical treatment methods including, but not limited to, tissue bulking, filling voids, forming occlusions, absorbing fluids, and delivering medications.
Monomer and Crosslinking Agent
Monomers that may be used in the present process for preparing microspheres are water miscible monomers including, but not limited to, acrylic acid, methacrylic acid, salts (such as sodium and ammonium) of acrylic acid and methacrylic acid, acrylamide, methacrylamide, N-substituted acrylamides, N-substituted methacrylamides, 2-acryloylethane-sulfonic acid, 2-methacryloylethane-sulfonic acid, salts of 2-acryloylethane-sulfonic acid and 2-methacryloylethane-sulfonic acid, styrene-sulfonic acid, salts of styrene-sulfonic acid, 2-hydroxyethyl acrylate, and 2-hydroxyethyl methacrylate. Monomers may be used singly or in combinations as co-monomers. Monomers that perform well as single monomer components (subgroup 1) include acrylic acid, methacrylic acid, salts (such as sodium and ammonium) of acrylic acid and methacrylic acid, 2-acryloylethane-sulfonic acid, 2-methacryloylethane-sulfonic acid, salts of 2-acryloylethane-sulfonic acid and 2-methacryloylethane-sulfonic acid, styrene-sulfonic acid, and salts of styrene-sulfonic acid. Preferably, the following monomers are used as co-monomers with at least one of the monomers from subgroup 1: acrylamide, methacrylamide, N-substituted acrylamides, N-substituted methacrylamides, 2-hydroxyethyl acrylate, and 2-hydroxyethyl methacrylate. Most useful in producing microspheres for medical applications are monomers having biocompatibility such as acrylic acid, methacrylic acid, salts of acrylic acid and methacrylic acid, 2-hydroxyethyl acrylate and 2-hydroxyethyl methacrylate, and combinations thereof. In one embodiment the monomer is a combination comprising acrylic acid and at least one monomer from the group of sodium acrylate, 2-hydroxyethyl methacrylate, 2-hydroxyethyl acrylate, styrene sulfonic acid, and the sodium salt of styrene sulfonic acid. In another embodiment, the monomer is styrene sulfonic acid or a combination comprising styrene sulfonic acid and the sodium salt of styrene sulfonic acid.
Many of these monomers are liquids which are miscible with water. For monomers that are solids, an aqueous solution of the monomer may be prepared, and this monomer solution is miscible with water. Acid monomers and salts of monomers may be combined to adjust the pH of a monomer solution. It is particularly useful to partially neutralize an acid monomer, thereby providing a mixture of acid monomer and monomer salt. Acid monomers that may be used are, for example, acrylic acid, methacrylic acid, 2-acryloylethane-sulfonic acid, 2-methacryloylethane-sulfonic acid, styrene-sulfonic acid, and combinations thereof. A monomer prior to partial neutralization is referred to as an initial monomer. An acid monomer is typically partially neutralized using a base. Suitable bases include, but are not limited to, sodium hydroxide, potassium hydroxide, ammonium hydroxide, lithium hydroxide and combinations thereof. Bases containing divalent cations, such as calcium hydroxide and barium hydroxide may also be used; however, they are preferably used in combination with a base containing monovalent cations because divalent cations have a strong tendency to induce ionic crosslinking, which could severely alter the desirable properties of the microspheres. For some applications it may be desirable to substitute a portion of the base with barium hydroxide (Ba(OH)2) to introduce a radio-opaque element, which makes the resulting microspheres amenable to x-ray imaging. Barium hydroxide may be used in a ratio of up to about 1:1 by weight of Ba(OH)2 to NaOH, to produce a combination salt that includes barium salt. Alternatively, a barium monomer salt may be included in a monomer combination.
A crosslinking agent that is miscible with an aqueous monomer solution is copolymerized with the monomer in the present process. Examples of crosslinking agents that may be used include, but are not limited to, N,N′-methylene-bis-acrylamide, N,N′-methylene-bis-methacrylamide, N-methylolacrylamide, N-methylolmethacrylamide, glycidyl acrylate, glycidyl methacrylate, polyethylene glycol diacrylate and polyethylene glycol dimethacrylate (which are most useful with hydrophobic monomers), polyvalent metal salts of acrylic acid and methacrylic acid, divinyl benzene phosphoacrylates, divinylbenzene, divinylphenylphosphine, divinyl sulfone, 1,3-divinyltetramethyldisiloxane, 3,9-divinyl-2,4,8,10-tetraoxaspiro[5,5]undecane, phosphomethacrylates, and polyol polyglycidyl ethers such as ethylene glycol diglycidyl ether, glycerin triglycidyl ether, glycerin diglycidyl ether, and polyethylene glycol diglycidyl ether, and combinations thereof. The amount of crosslinking agent included for copolymerization may vary and is inversely related to the amount of swell capacity in the microspheres produced using the present process. Different amounts of crosslinking agent result in swelling capacities over a range of about 1.5 grams of water per gram of microspheres to over 100 grams of water per gram of microspheres. Generally useful is an amount of crosslinking agent that results in microspheres with a swell capacity of at least about 50 grams of water per gram of microspheres. Particularly useful is an amount of crosslinking agent that results in microspheres with a swell capacity of at least about 70 grams of water per gram of microspheres. The exact amount of crosslinking agent needed will vary depending on the specific agent used and can be readily determined by one skilled in the art. The amount of crosslinking agent is calculated as Mol % (mole percent) based on the sum of the moles of monomer and moles of crosslinking agent. Thus, the Mol % is calculated as moles of crosslinking agent/(moles of monomer+moles of crosslinking agent). For example, 4.0 Mol % of N,N′-methylenebisacrylamide with respect to moles of acrylic acid monomer+sodium acrylate+crosslinking agent produces microspheres with a swell of about 50 grams of water per gram of microspheres, 2.9 Mol % of N,N′-methylenebisacrylamide produces microspheres with a swell of about 70 grams of water per gram of microspheres, and 2.3 Mol % of N,N′-methylenebisacrylamide produces microspheres with a swell of about 107 grams of water per gram of microspheres. Preferably, the Mol % of crosslinking agent is equal to or less than about 5 Mol %, preferably, equal to or less than about 4 Mol %, more preferably about 0.08 Mol % to about 4 Mol %, most preferably about 0.08 Mol % to about 2.3 Mol % relative to total moles of monomer and crosslinking agent. Microspheres with very high swell (i.e., over 250 grams of water per gram of microspheres) can be prepared using a hydrophilic monomer such as sodium acrylate, a low amount of crosslinking agent (e.g., 0.083 Mol % of N,N′-methylenebisacrylamide), with low temperature drying conditions, as described in Example 35 below.
First Solution
A monomer and crosslinking agent as described above are prepared in an aqueous solution, together with additional components, which is herein called the “first solution”. The monomer is generally included at about 0.5% to about 30% as weight percent of the first solution. Monomer weight percents of about 15% to about 25% and about 20% to about 25% are particularly useful in the process of the invention. If a combination of monomers is used in the process, the total amount of all the monomers is about 0.5% to about 30%, in addition from about 15% to about 25%, and in addition from about 20% to about 25%, as weight percent of the first solution.
The pH of the first solution may vary and is a factor in the swell capacity of the microspheres prepared in the process of the invention. The useful pH range of the first solution also depends on the particular monomer or combination of monomers used. If the first solution contains at least one monomer from subgroup 2 consisting of acrylic acid, methacrylic acid, salts of acrylic acid and methacrylic acid, acrylamide, methacrylamide, N-substituted acrylamides, N-substituted methacrylamides, 2-hydroxyethyl acrylate, and 2-hydroxyethyl methacrylate, but does not contain a monomer from subgroup 3 consisting of 2-acryloylethane-sulfonic acid, 2-methacryloylethane-sulfonic acid, salts of 2-acryloylethane-sulfonic acid and 2-methacryloylethane-sulfonic acid, styrene-sulfonic acid, and salts of styrene-sulfonic acid, then the pH of the first solution is at least about 3, preferably between about 3.5 and about 10, more preferably between about 5 and about 9, to produce microspheres with a high swell capacity. For example, a mixture of acrylic acid and sodium acrylate at a pH of between about 3.5 and about 10, and a 2 to 5 Mol % of N,N′-methylenebisacrylamide crosslinking agent (with respect to the monomer), when used in the process of the invention, produces microspheres with a swell capacity of at least about 80 grams of water per gram of microspheres. If the first solution contains at least one monomer from subgroup 3 consisting of 2-acryloylethane-sulfonic acid, 2-methacryloylethane-sulfonic acid, salts of 2-acryloylethane-sulfonic acid and 2-methacryloylethane-sulfonic acid, styrene-sulfonic acid, and salts of styrene-sulfonic acid, then the pH of the first solution is less than about 3 to produce highly swellable microspheres (see Examples 36-38).
The pH of the first solution may be adjusted in any number of ways. For example, if the monomer is prepared as a monomer solution, as described above, the pH of the monomer solution will govern the pH of the first solution. In the case of an acid monomer, the pH of the monomer solution is related to the amount of base or monomer salt added to the acidic monomer solution. Alternatively, the pH of the first solution may be adjusted as required by the addition of acid or base after all the components have been added.
Included in the “first solution” is a component that can modify the viscosity of an aqueous solution to provide a surface tension that allows droplet formation in the aqueous/organic suspension that is formed during the present microsphere preparation process. This component is referred to herein as a “protecting colloid”. A variety of natural and synthetic compounds that are soluble in aqueous media may be used as a protecting colloid including cellulose derivatives, polyacrylates (such as polyacrylic acid and polymethacrylic acid), polyalkylene glycols such as polyethylene glycol, partially hydrolyzed polyvinyl alcohol and other polyols, guar gum, and agar gum. Particularly useful are cellulose ethers such as methyl cellulose, hydroxymethyl cellulose, hydroxyethyl cellulose, ethylhydroxyethyl cellulose, hydroxypropyl cellulose, ethyl cellulose, and benzyl cellulose; as well as cellulose esters such as cellulose acetate, cellulose butylate, cellulose acetate butylate, cellulose propionate, cellulose butyrate, cellulose acetate propionate, cellulose acetate butyrate, and cellulose acetate phthalate. The amount of the protecting colloid in the first solution is sufficient to reduce microdroplet coalescence in the aqueous/organic suspension, and is generally between about 0.1% and about 3% by weight % of the first solution. Preferred is methyl cellulose at about 0.5% to about 0.6% by weight.
An emulsifier is included in the first solution to promote the formation of a stable emulsion on addition of the first solution to an organic second solution (described below). Any emulsifier which stabilizes the aqueous/organic emulsion may be used. Suitable emulsifiers include, but are not limited to, alkylaryl polyether alcohols such as the Triton™ X nonionic surfactants commercially available from Union Carbide (Danbury, Conn.). These products generally contain mixtures of polyoxyethylene chain lengths and include, for example, Triton® X-100: polyoxyethylene(10) isooctylphenyl ether; Triton® X-100, reduced: polyoxyethylene(10) isooctylcyclohexyl ether; Triton® N-101, reduced: polyoxyethylene branched nonylcyclohexyl ether; Triton® X-114: (1,1,3,3-tetramethylbutyl)phenyl-polyethylene glycol; Triton® X-114, reduced: polyoxyethylene(8) isooctylcyclohexyl ether; Triton® X-405, reduced: polyoxyethylene(40) isooctylcyclohexyl ether; and Triton™ X-405: polyoxyethylene (40) isooctylphenyl ether, 70% solution in water. Particularly suitable is Triton™ X-405, 70 wt % solution, which is an alkylaryl polyether alcohol preparation having an average of at least about 30 ethylene oxide units per ether side chain. Typically, the emulsifier in the first solution is used at a concentration of about 1% to about 10% by weight % of the first solution.
In addition, the first solution includes a polymerization initiator. The initiator used in the process of the invention is a water soluble azo initiator which has a low temperature of activation. Azo initiators are substituted diazo compounds that thermally decompose to generate free radicals and nitrogen gas. The temperature of activation of the azo initiator used is low enough so that the boiling point of an organic second solution (described below) is above the azo initiator activation temperature. Examples of suitable low temperature water soluble azo initiators include, but are not limited to, 2,2′-azobis(2-amidinopropane)dihydrochloride; 4,4′-azobis(4-cyanopentanoic acid); and 2,2′-azobis(2-[2-imidazolin-2-yl])propane dihydrochloride. A particular azo initiator, having a particular activation temperature, is used with an organic second solution composition (described below) at a temperature and with a reaction time period that is effective in initiating polymerization. Most effective is use of an azo initiator at a temperature that is close to its optimal activation temperature and which is also below the boiling temperature of the organic second solution. However, an azo initiator may be used at a temperature that is lower than its optimal activation temperature in order to stay below the boiling temperature of the organic second solution, but this will require a longer reaction time for polymerization. A particularly suitable azo initiator has an activation temperature that is less than about 53° C. and this azo initiator is used with an organic second solution having a boiling temperature of about 55° C. A particularly suitable azo initiator is VA-044™ (2,2′-azobis(2-[2-imidazolin-2-yl])propane dihydrochloride, commercially available from Wako Pure Chemical Industries, Ltd., Richmond, Va.) having an activation temperature of between 51° C. and 52° C.
The azo initiator has advantages over other initiators such as persulfates and hydroperoxides. The azo initiator is effective when used in very low amounts, in contrast to other initiators. The azo initiator is used at about 0.1% to 1.0% by weight % of monomer. Preferably about 0.5% azo initiator is used. The low level of azo initiator results in very low levels of initiator contamination in the polymerized hydrogel as compared to contamination resulting from use of other initiators. In addition, there is no metal contamination resulting from the azo initiator, while other initiators typically include metal catalysts that do leave metal contamination in the polymerized product. In addition, other typical initiators are sensitive to oxygen, and, therefore, solutions in contact with these initiators must be de-aerated. The remaining oxygen content of the de-aerated solutions is variable, leading to inconsistency in the microsphere forming process. With use of an azo initiator, no de-aeration is required, which reduces the complexity of solution preparation for use in the microsphere formation process and increases the consistency of microsphere preparation. In addition persulfate initiators generally give more inconsistent conversion and yields of microspheres than azo initiators.
Second Solution
An organic solution acts as a dispersion medium in the process of microsphere preparation, and is herein called the “second solution”. The second solution comprises at least one substantially chlorinated hydrocarbon of less than 6 carbon units, excluding halogenated aromatic hydrocarbons. A substantially chlorinated hydrocarbon may be a hydrocarbon that is at least 50% chlorinated, as well as a fully chlorinated hydrocarbon. Particularly suitable is a chlorinated solvent that readily dissolves ethyl cellulose to a homogeneous solution, boils above at least about 50° C. and has a density able to support microsphere formation in aqueous/organic suspension. A particularly useful organic medium in the process of microsphere preparation is a mixture containing chloroform and methylene chloride. Methylene chloride alone does not have a high enough boiling temperature to allow the use of a low temperature aqueous azo initiator. Chloroform alone is not sufficient to support microsphere formation. The combination of chloroform and methylene chloride provides an organic solution which has a boiling temperature allowing use of a low temperature aqueous azo initiator and which supports microsphere formation in the aqueous/organic suspension. Chloroform and methylene chloride may be used in volume ratios between about 20:1 and about 1:20. More suitable is a chloroform and methylene chloride solution with a volume ratio between about 5:1 and 1:5. Particularly suitable is a volume ratio of 3:1 chloroform:methylene chloride solution which has a boiling temperature of about 53° C.
Additionally, other solvents or solvent mixtures may be used in combination with a substantially chlorinated hydrocarbon such as methylene chloride. For example, it may be desirable to substitute for chloroform in the chloroform-methylene chloride mixtures described above because of the health hazards of chloroform. Suitable solvent or solvent mixtures to substitute for chloroform may be selected by matching the Hansen solubility parameters (Hansen, Hansen Solubility Parameters, A User's Handbook, CRC Press LLC, Boca Raton, Fla., 2000) of particular solvent or solvent mixtures to those of chloroform. The Hansen solubility parameters are an extension of the Hildebrand solubility parameters. According to Hansen, “the basis for the Hansen Solubility Parameters (HSP) is that the total energy of vaporization of a liquid consists of several individual parts, that arrive from (atomic) dispersion forces, (molecular) permanent dipole-permanent dipole forces and (molecular) hydrogen bonding (electron exchange).” Materials having similar HSP have high affinity for each other. The basic equation for the HSP is that the total cohesion energy, E, must be the sum of the individual energies:
E=ED+EP+EH
Where ED is the Hansen dispersion cohesion energy, Ep is the Hansen polarity cohesion energy, and EH is the Hansen hydrogen bonding cohesion energy. Dividing this expression by the molar volume, gives the total Hildebrand solubility parameter as the sum of the squares of the Hansen components:
δ2=δD2+δP2+δH2
Chloroform has a Hansen dispersion of 17.8, Hansen polarity of 3.1 and Hansen hydrogen bonding of 5.7 in units of the square root of megapascals (mPa1/2). A software program (Molecular Modeling Pro Plus, ChemSW, Fairfield, Calif.) is available to calculate the Hansen solubility parameters from molecular structure. Preferred solvent mixtures have a sum of the differences (in absolute value) in Hansen solubility parameters relative to the Hansen solubility parameters of chloroform of less than about 0.21. A sample calculation of the sum of the differences in Hansen solubility parameters for a mixture of 30 vol % (percent by volume) ethyl heptanoate and 70 vol % phenethyl acetate relative to chloroform is shown in Table A. Suitable solvent mixtures are given in Table B.
In one embodiment, the second solution comprises a combination of a solvent mixture of 30 vol % ethyl heptanoate (CAS No. 106-30-9) and 70 vol % phenethyl acetate (CAS No. 103-45-7), with methylene chloride in a volume ratio of about 20:1 to about 1:20, in addition about 5:1 to about 1:5, and further in addition of about 3:1.
The second solution also comprises a viscosity modifying component that provides a surface tension that allows droplet formation in the aqueous/organic suspension formed during the present microsphere preparation process. This viscosity modifying component is again called a “protecting colloid”. A variety of natural and synthetic compounds soluble in organic media may be used as a protecting colloid, including, but not limited to, cellulose derivatives, polyacrylates (such as polyacrylic acid and polymethacrylic acid), polyalkylene glycols such as polyethylene glycol, partially hydrolyzed polyvinyl alcohol and other polyols, guar gum, and agar gum. Particularly useful are cellulose ethers such as methyl cellulose, hydroxymethyl cellulose, hydroxyethyl cellulose, ethylhydroxyethyl cellulose, hydroxypropyl cellulose, ethyl cellulose, and benzyl cellulose; as well as cellulose esters such as cellulose acetate, cellulose butylate, cellulose acetate butylate, cellulose propionate, cellulose butyrate, cellulose acetate propionate, cellulose acetate butyrate, and cellulose acetate phthalate. The amount of the protecting colloid in the organic second solution is sufficient to reduce microdroplet coalescence in the aqueous/organic suspension, and is generally between about 0.5% and about 5% by weight % of the organic second solution. Particularly suitable is ethyl cellulose at about 1.5% by weight.
Process for Microsphere Preparation
The first solution and the second solution are combined with agitation to form a first suspension. The second solution is used in an amount that is adequate to form a good suspension, while the amount may be as great as is practical. Generally the volume ratio of second to first solutions is in the range of about 10:1 to about 2:1. Preferably the volume ratio of second to first solutions is in the range of about 6:1 to about 4:1.
The first and second solutions may be combined in any order. Specifically, the first solution can be added to the second solution, the second solution can be added to the first solution, or the two solutions can be combined simultaneously. Preferably, the first solution is added to the second solution. During the combination of the first and second solutions, the resulting mixture is agitated at a rate capable of forming a uniform suspension from the two solutions. Agitation may be by any method which thoroughly mixes the two solutions, such as shaking or stirring. Typically, the second solution is stirred in a container while the first solution is poured into the same container. The combined first and second solution is agitated at a temperature that is below the azo initiation temperature (and above the freezing point of the solution) to form a uniform, first suspension. Generally the temperature is below about 50° C., and more typically is below about 40° C. A temperature that is below about 30° C. is preferred. Typically the first suspension is stirred at about 100 to 600 rpm, depending on the size of the container, at room temperature for about one-half to one hour.
The agitation of the first suspension allows formation of small droplets in the suspension. The size of the forming droplets, and therefore the size of the microspheres that are produced, is related to the rate of agitation. As the agitation is reduced, droplets coalesce. Agitation is maintained at a rate sufficient to reduce droplet coalescence allowing the formation of micron sized microspheres. For example, for the formation of microspheres in the size range of 40 to 500 microns, stirring is typically about 150-250 rpm when using a one liter container. The optimum agitation rate for any particular system will depend on many factors, including the particular monomer, crosslinking agent, and solvent system used, the geometry of the container, the geometry of the agitator, and the desired microsphere properties for the intended application. For example, the size of the microspheres depends on the agitation rate. In general, larger microspheres are obtained at lower agitation rates. The agitation rate for any given conditions can be readily optimized by one skilled in the art using routine experimentation.
After the formation of the first suspension, a low level of heat is applied such that the temperature of the first suspension is brought to a temperature that is below the boiling temperature of the first solution, and below or at the boiling temperature of the second solution. Typically the temperature is between about 50° C. and 55° C., depending on the mixture of the second solution. It is preferred to bring the temperature of the first suspension made with a chloroform and methylene chloride ratio of about 3:1 to about 51° C. to 52° C. At this temperature the low temperature azo initiator is activated. The first suspension is agitated until it forms a second suspension comprising a precipitate of gelatinous microspheres in the suspending medium, which is predominantly an organic liquid phase. The gelatinous precipitate appears as a milky material which falls out of the suspension. Additionally, a white foam may be seen on top of the second suspension. Typically stirring of the first suspension to form the second suspension at the elevated temperature is for about 8-10 hours. The second suspension is agitated for another period of time at room temperature to ensure that the polymerization and microsphere formation is complete. During this time the second suspension cools to a temperature which is easily handled. Generally this is at or below about 30° C. Room temperature, typically at about 25° C., is conveniently used. Typically stirring remains at about 150-250 rpm, when using a one liter container, for about 8-14 hours.
Agitation is ceased, allowing the formed microspheres to settle to the bottom of the container. Removing the water from these hydrogel microspheres may be accomplished by washing with a dehydrating solvent such as methanol, ethanol, or acetone. Particularly useful is methanol, which is added, and the mixture is optionally agitated gently for about an hour to allow good solvent exchange. The microspheres are then recovered by a method such as by decanting or filtering, and may be washed a second time with methanol and again recovered. With removal of the water, the microspheres change in appearance from milky and gelatinous to hard and opaque white. The microspheres finally may be washed in ethanol, which is desirable for removal of residual methanol, particularly for microsphere use in medical applications. The washed microspheres in ethanol form one type of microsphere slurry. The microspheres optionally may be dried to form a powder of microspheres. Drying rids the microspheres of remaining washing solvent and additional water. Drying may be by any standard method such as using air, heat, and/or vacuum. Particularly useful is drying under vacuum in a vacuum oven set at about 20° C. to about 100° C. with a nitrogen purge. The use of lower drying temperatures requires longer drying times. For preparation of highly swellable microspheres, drying at room temperature (i.e., about 20° C. to about 25° C.) under vacuum with a nitrogen purge is preferred (see Example 34). A small amount of water generally remains in the microspheres after drying. The amount of remaining water may be about 1% to 10% of the microsphere total weight. The resulting microsphere preparation, though retaining a small amount of water in the microspheres, flows when tilted or swirled in a container and thus forms a free-flowing microsphere powder.
Microsphere Physical Properties
Microspheres prepared according to the present process are substantially spherical. A population of microspheres has sphericity measurements centered near about 95%, within a range of about 80% to about 100%. The population may include some individual microspheres which have a lower sphericity measurement, while maintaining the high sphericity measurement for the population as a whole.
The microspheres are in the size range of about 10 to about 730 microns in diameter, in addition from about 14 to about 730 microns in diameter. A prevalence of the microspheres are in the size range of about 25 to about 250 microns in diameter, as seen when analyzing a small sample size of microspheres. A heterogeneous size mixture of microspheres may be separated into microsphere samples of specific size ranges, if desired, for specific applications. Microspheres may be separated by methods such as fluidized bed separation and sieving, also called screen filtering. Particularly useful is sieving through a series of sieves appropriate for recovering samples containing microspheres of desired sizes. For example, separate samples of microspheres may be obtained using a series of sieves with mesh sizes of 35 to 400 microns. Separate microsphere samples may be obtained that have diameters ranging between about 30 and about 44 microns; about 115 and about 165 microns; about 180 and about 330 microns; and with size ranges also falling between and outside of these exemplary groups. These samples of size separated microspheres exemplify the production of microsphere preparations having a predominant size ranging between about 30 microns and 600 microns in diameter and including microspheres in a size range that is generally within +/−30% of the median for about 90% of the sample. Microsphere preparations may be produced having microspheres in a size range that is generally within +/−20% of the median for about 90% of the sample.
The microspheres prepared according to the present process have a high density, yet a high capacity for swell. The microspheres have low porosity, especially as compared to the microspheres described in U.S. Pat. No. 6,218,440, as viewed by scanning electron microscopy (SEM). The microspheres of U.S. Pat. No. 6,218,440 have cavities joined by interconnecting pores wherein at least some of the cavities at the interior of the material communicate with the surface of the material. These microspheres have pores throughout and a rough porous surface as well. The microspheres prepared by the present process have by comparison a relatively small number of voids embedded within a solid material. Generally, although not invariably, these voids are closed cell voids that are not interconnected to each other or to the surface of the microsphere. The surface of the present microspheres is generally smooth and rounded, although some surface imperfections may be present. The porosity of microspheres can also be assessed by density measurements. One preparation of microspheres prepared by the present process had a bulk density of 0.68 g/cm3 (Example 6). It is expected that microspheres produced by the present process will have a bulk density of at least about 0.5 g/cm3. In contrast, the bulk density of microspheres prepared according to one method in the prior art (U.S. Pat. No. 6,218,440 Example 2) was measured to be 0.182 g/cm3 (see present Example 6). Individual microsphere density of the present microspheres is between about 0.9 g/cm3 and about 2 g/cm3, while the individual density of microspheres prepared according to one method in the prior art (U.S. Pat. No. 6,218,440 Example 2) was measured to be 0.8 g/cm3 or less (see present Example 6).
The density, and porosity, of microspheres play a role in the durability of the microspheres. The microspheres prepared by the present process are highly durable in that the swelled microspheres have substantial resistance to fracture as they are passed through a small bore needle. Under the same conditions, swelled highly porous microspheres do fracture, and thus have low durability. For example, microspheres prepared by the present process that are swelled and passed through a 20 gauge needle maintain an average diameter similar to that of the starting sample, while the average diameter of microspheres prepared according to one method in the prior art (U.S. Pat. No. 6,218,440 Example 2) after passing through a 20 gauge needle is reduced by almost half indicating fracture of the particles.
As described above, the swell capacity (amount of water uptake) of microspheres prepared by the present process may vary depending on the amount of crosslinking agent added to the first solution. For example, crosslinking agent may be added in such an amount as to impart a swell capacity to the microspheres of about 50 grams of water per gram of microspheres, an amount to impart a swell capacity of about 70 grams per gram of microspheres, and alternatively an amount to impart a swell capacity of about 100 grams per gram of microspheres. Particularly suitable is a microsphere preparation having at least 70 grams of water uptake per gram of microsphere powder. Using the same amount of crosslinking agent, microspheres prepared according to one method in the prior art (U.S. Pat. No. 6,218,440 Example 2) had less than half of this swell capacity (see Example 6, Table 7).
The microspheres made by the present process exhibit rapid swell. Individual microspheres can be seen to reach a maximum size within about 15 seconds of contacting the microspheres with water. Thus the individual microspheres reach their full swell capacity within about 15 seconds, and typically within about 10 seconds. A population of microspheres also has rapid swell as long as each microsphere has sufficient exposure to water. In general, when contacting a population of microspheres with water, those microspheres in the center of the population, or on the bottom of a container, do not have full exposure to water so that their swell time is longer. For example 1 gram of microspheres may reach 50% of full swell in 5 seconds and about 70% of full swell in 10 seconds with water exposure as described in the General Methods. Generally full swell is reached within 30 seconds for a population of microspheres under the described water contact conditions.
An additional attribute of the microspheres prepared by the present process is the capacity to deform following swell. When placed under pressure, the swelled microspheres do not maintain their substantially spherical shape, but compress in the axis of the pressure and expand in the axis that is perpendicular to the pressure. Thus environmental factors, such as pressure of a flowing medium or from the walls of an enclosing container, may cause deformation of the microspheres. In addition, pressure of individual microspheres next to each other may cause deformation. This ability to deform is thought to be imparted and enhanced through the closed cell void structure of the microspheres. While not wishing to be bound by theory, it is thought that the closed cell voids are able to compress allowing the swelled hydrogel in the microspheres to deform maximally.
This ability to deform allows the microspheres to take on a shape of a containing space, and to fill that space. Additionally, deformed microspheres have increased surface area contact with each other, as compared to the contact area between spherical beads. The increased surface area contact between the deformed microspheres provides a more compact structure than is achievable with non-deforming spherical microspheres. This compact structure provides high resistance to penetration. The deformability is highly desirable in some applications such as in embolization treatment, where the deformed, compact microspheres may provide strong blockage at target vascular sites. In a test system that uses a flexible, substantially nonexpendable tubing having an internal diameter of 1.58 mm, the swelled, deformed microspheres were able to form occlusions that withstood very high pressures. For example, 15 mg of dry microspheres, when fully swelled, formed microsphere occlusions that were not dislodged by water pressure less than about 114 mm Hg (15.2 kilopascals (kPa)). Starting with 18 mg of dry microspheres the occlusions formed were dislodged by water pressure at about 570 mm Hg (76.0 kPa), and starting with 20 mg of dry microspheres the occlusions formed withstood over 1,000 mm Hg of pressure (133 kPa).
Microsphere Properties Advantageous for Medical Applications
Microspheres prepared by the present process are biocompatible in that they lack cytotoxicity, are non-inflammatory, and are non-hemolytic. The microspheres have a swell response in whole blood that is similar to the swell response in water: achieving up to 100-fold swell within seconds. These properties allow the microspheres described herein to be used in medical applications, where advantage may be taken of their full swell potential. In addition, the resistance to fracture of the microspheres described herein makes them particularly suitable for medical applications since resistance to fracture reduces the potential for effects such as occlusion downstream of the target site, unwanted inflammatory response, exacerbation of clotting cascade, and loss of therapeutic occlusion.
The Microsphere Preparation
The microsphere preparation of the present invention is prepared according to the present process and contains swellable/deformable microspheres having the properties described herein. The microsphere preparation may be the direct product of the process prior to drying, where the microspheres form a microsphere slurry including extraction solvent. The additional drying step of the process produces a microsphere powder. The microsphere powder may be made available for use as a powder or for addition of a liquid appropriate to the intended use. Addition of a liquid to the microsphere powder produces a microsphere slurry or microsphere suspension. Liquids used in a microsphere suspension may be any that are appropriate for the intended use. For example, a biocompatible liquid that controls swell is used to suspend microspheres for medical uses, such as tissue augmentation, wound treatment, and embolization. Typical swell-control biocompatible liquids include, for example, propylene glycol, dimethylsulfoxide (DMSO), Ethiodol®, MD-76®, and mineral oil. Ethiodol® and MD-76® are contrast agents typically used in medical intravascular arteriography or lymphography procedures. Ethiodol® contains iodine organically combined with ethyl esters of the fatty acids of poppyseed oil and is available from SAVAGE Laboratories® (Melville, N.Y.). MD-76® is an aqueous solution of diatrizoate meglumine (CAS No. 131-49-7, 66 wt %) and diatrizoate sodium (CAS No. 737-31-5, 10 wt %) buffered with monobasic sodium, with a pH of 6.5 to 7.7, having organically bound iodide to provide for radiological visualization. MD-76® is manufactured by Mallinckrodt Inc. (St. Louis, Mo.).
Microsphere Suspension
A microsphere preparation made according to the present process is used to prepare a suspension for medical treatment, herein called a “microsphere suspension”. Sterility is an important factor in medical treatment. The described microsphere preparation process including a final ethanol wash, provides a sterilization treatment. Further sterilization may be performed by extending the ethanol wash for a long period of time, such as overnight. Sterility may be enhanced by using additional measures such as carrying out the process for making the microspheres in a sterile environment, and treating the microsphere preparation with UV light, ethylene oxide or gamma radiation, as is known to one skilled in the art.
The microsphere suspension includes a biocompatible carrier. The carrier provides not only a medium to suspend and administer the microspheres, but also to control the swelling of the microspheres. A carrier used in the suspension may have a low enough viscosity to allow delivery of the microspheres through small-bore needles and catheters, such as those of 20 gauge or 7 French (F) or smaller. A gauge measurement is used for needles, while a French measurement is used for catheters, both of which designate the outside diameter. The inside diameter of a needle or catheter is related to the outside diameter, but also depends on the thickness of the wall and so can vary between manufacturers. Thus precise measurements of the inside diameters of needles and catheters are not specified by the gauge or French unit. However, inside bore diameters of specific catheters and needles are known or can readily be obtained by one skilled in the art. Biocompatible carriers that limit swell of the microspheres, and thus are swell-control media, include the commonly used contrast agents Ethiodol® (SAVAGE Laboratories®, Melville, N.Y.) and MD-76® (Mallinckrodt Inc., St. Louis, Mo.). In addition, swell may be controlled by salt concentration and ionic strength, as well as with pH. The organic polar solvent dimethylsulfoxide (DMSO) was found, as described in the Examples herein, to be a useful medium for controlling microsphere swelling, and for making a microsphere suspension for administering the microspheres. Microspheres suspended in DMSO at concentrations between about 60% and 100% undergo appreciably no swell. Particularly suitable biocompatible carriers are those containing DMSO above about 60% concentration, those with an acidic pH, and contrast agents. The contrast agent MD-76® allows some swell, ranging between about 3.5× and about 7.5× the original volume, and may be used as a swell-control medium. Different carriers may be mixed, such as combining a percentage of DMSO and a contrast agent to establish the desired amount of microsphere swell (explained below) in the microsphere suspension.
The microsphere concentration in the microsphere suspension varies depending on the carrier used and the size catheter or syringe to be used for administering the suspension, which in turn depends on the specific treatment to be carried out. In addition, the size of the microspheres affects the concentration used, where samples of different sized microspheres may be prepared, for example by sieving, as described herein. For example, 250 mg/mL concentration of approximately 250 micron microspheres in DMSO (no swell) may be used with catheters of 6F and larger. For delivery of high concentrations of microspheres with smaller catheters, such as 5F and smaller, it may be desirable to have limited swell of the microspheres for administering the microsphere suspension. The limited swell may take place prior to or during the administering. The limited swell may be up to about 10× the original volume of the microspheres. Limited swell provides deformability of the microspheres which allows them to pass through small diameter catheters and needles. Limited swell may be achieved by methods such as adjusting the salt concentration, pH or DMSO concentration of the carrier, or with use of a contrast agent. For example, with about 50% or less DMSO concentration in the carrier, the microspheres begin to swell. In addition, the microspheres may swell to between about 3.5× and 7.5× the original volume in contrast agent. Passage through 5F catheters may be achieved with suspensions containing, for example, 150 mg/mL of 250 micron microspheres in MD-76®. Also, microsphere suspensions containing 300 mg/mL of 50-150 micron microspheres in MD-76® can pass through a 5F catheter. The specific size and concentration of microspheres, as well as the desired carrier, may be chosen by one skilled in the art for the particular medical treatment to be performed.
Medical Treatments
A variety of medical treatments may be accomplished by administering microspheres, prepared using the present process, to a mammal. Such medical treatments may involve void filling, tissue bulking, occlusion, fluid absorption, and/or delivery of medication. For treatments such as void filling, tissue bulking and occlusion, typically a microsphere suspension is administered to a target anatomical site using a needle or a catheter. The concentration of the suspension and the size of needle or catheter for delivery is determined by the target site location, and can readily be determined by one of skill in the art.
Forming occlusions in non-vasculature locations may be used as treatments for blocking flow of bodily fluids other than blood. Microspheres made by the present process may be administered in any medical situation where forming an occlusion in a passageway provides a desirable treatment. Target sites that are non-vasculature anatomical passageways include, for example, tubes and ducts. The properties of the microspheres prepared by the present method, such as the particularly durable occlusions they form, as described above, make them valuable for occlusion treatments. An example is occlusion of urinary tract fistulas. Fistulas of the urinary tract are abnormal passages connecting the urinary tract to other organs, including the skin and vagina (Avritscher et al. (2004) Radiographics 24 Suppl 1:S217-236). Urinary tract fistulas can occur spontaneously or as a result of pelvic surgery. When present, urinary tract fistulas lead to urine leakage. A remedy for this situation is to occlude individual fistulas, or to occlude the ureter and divert urine flow using an implanted nephrostomy tube. A suspension of microspheres, prepared using the present process, may be introduced into the urinary tract using a catheter. The microspheres swell upon exposure to physiological fluids forming an occlusion that completely blocks flow of urine through the fistula or ureter. Occlusion may also be used in treatment of pancreatitis (Cavouti et al. (1988) Surgery 103(3):361-366). Pancreatitis is inflammation of the pancreas, in which pancreatic enzymes digest the pancreatic tissue. Pancreatitis may result from situations such as excess alcohol consumption or gallstones, and often leads to severe pain, nausea, and vomiting. A treatment for severe chronic pancreatitis is occlusion of the pancreatic duct. A suspension of microspheres, prepared using the present process, may be introduced into the pancreatic duct using a catheter, while the pancreatic duct is visualized endoscopically. The microspheres swell upon exposure to physiological fluids forming an occlusion that blocks the pancreatic duct.
Birth control may be attained by occlusion in both males and females (Chvapil et al. (1990) Journal of Reproductive Medicine 35(9):905-910; Davis et al. (1979) Obstetrics & Gynecology 1979; 53:527-529). Introduction of microspheres, prepared in the present process, into the vas deferens may be performed to create an occlusion, thereby blocking flow of seminal fluid. Introduction of microspheres, prepared in the present process, into the fallopian tube may be performed to create an occlusion, thereby blocking the entry of eggs into the uterus. In both cases the microspheres may be introduced using catheters.
An additional example of an occlusion treatment is in the case of dry eyes (Hamano (2005) Seminars in Ophthalmology 20(2):71-74). Dry eyes result from insufficient tear production or poor tear quality. One remedy for dry eyes is to occlude the punctum, which is the opening of the tear duct on the eyelid margin. The tear duct drains tears out of the eye, and occlusion of the punctum blocks tear drainage from the eye. When tear drainage from the eye is blocked, tears are kept in the eye for a longer period of time, and more tears are present to lubricate the eye. Microspheres prepared by the present process may be injected directly into the punctum, where they swell upon exposure to physiological fluids and form an occlusion, thereby providing a treatment for dry eye.
Tissue bulking is another medical treatment which may be used in a variety situations, and which benefits from the administering of microspheres prepared by the present method. Tissue bulking may be used as a treatment for disorders such as sphincter weakness, dermal scarring, loss of skin tone, gum degeneration, and other situations where there is thinning or degeneration of tissues. The rapid and high capacity swelling, deformability, as well as other characteristics of the present microspheres, make them an effective material for use in tissue bulking.
Microspheres prepared by the present method may be used as a bulking agent to treat gastro-esophageal reflux disease (GERD). GERD is the return of the stomach's acidic contents back up into the esophagus. In normal digestion, the lower esophageal sphincter opens to allow food to pass into the stomach and closes to prevent food and acidic stomach juices from flowing back into the esophagus. GERD occurs when the lower esophageal sphincter is weak or relaxes, allowing stomach contents to flow up into the esophagus. A treatment for this condition is to use a bulking agent to provide physical support to the lower esophageal sphincter (Ozawa et al. (2005) Annals of Thoracic and Cardiovascular Surgery 11(3):146-153). In this application, the present microspheres that are suspended in a biocompatible carrier are injected directly into the lower esophageal sphincter. The microspheres swell upon exposure to physiological fluids providing increased bulk at the injection site, which in turn provides the sphincter muscles with additional capability to control flow of digested food.
The microspheres prepared by the present method may also be used as a bulking agent to treat urinary incontinence, particularly, female stress urinary incontinence. Stress urinary incontinence is the loss of urine from the bladder caused by pressure occurring during activities such as exercise, coughing, and sneezing. One cause of this problem is the weakening of the urethral sphincter, a ring-shaped muscle at the base of the bladder that controls the flow of urine. A remedy for this condition is to use a bulking agent to provide physical support to the urethral sphincter (Madjar et al. (2003) Journal of Urology 170(6 Pt 1):2327-2329). In this application, the microspheres are suspended in a biocompatible carrier and are injected directly into the urinary sphincter. The microspheres swell upon exposure to physiological fluids providing increased bulk at the injection site, which in turn provides the sphincter muscles with additional capability to control urine flow.
Bulking of skin tissues, including fascia, subcutaneous and dermal tissues, may be used to treat skin disorders including scars, skin laxness, and skin thinning, and may be used in some types of cosmetic and reconstructive plastic surgery. Such disorders of the skin often are exhibited as contour deficiencies, which may be treated using the present microspheres. Contour deficiencies in the skin can occur as a result of factors such as aging, environmental exposures, weight loss, childbearing, surgery or disease. Contour deficiencies include frown lines, worry lines, wrinkles, crow's feet, marionette lines, stretch marks, and internal and external scars. Augmentation of the skin layers may reduce or eliminate contour deficiencies. The present microspheres, that are suspended in a biocompatible carrier, are injected into the desired skin layer where they swell upon exposure to physiological fluids. The swollen microspheres then augment the skin layer to modify the contour of the skin.
Medical situations may result in the occurrence of voids in either intra-organ or extra-organ locations, or there may be natural voids requiring treatment due to a medical condition. The rapid and high capacity swell, as well as deformability and other characteristics of the microspheres prepared by the present method make them desirable as a material to be used in void filling treatments. In intestinal diverticular disease, an intestinal diverticulum forms, which is a small outpouching in a weakened area of the bowel wall. When present, an intestinal diverticulum can become inflamed as a result of fecal trapping in the pouch, and can also hemorrhage. A treatment for this situation is to fill the outpouching thereby eliminating the trapping of material and reducing the risk of hemorrhage. The present microspheres may be introduced as a suspension into the intestinal diverticulum. Swelling of the microspheres upon contact with physiological fluids results in filling of the diverticulum.
Extra-organ voids may be formed following surgical excision of soft tissue or organs such as in the case of partial lung resections, hysterectomies, mastectomies, or bowel excisions. The vacant spaces that are created by removal of these organs fill with fluid and debris, creating mechanical compromise and elevating risk of infection. A treatment for this situation is to use a mechanical bulking agent to fill the extra-organ space (Giudicelli et al. (1979) Annales de Chirurgie 33(3):151-154). In this application, the microspheres are suspended in a biocompatible carrier, then injected into the extra-organ space (including the pleura, pericardium, or peritoneum) using a needle or catheter. The microspheres swell upon exposure to physiological fluids and fill the extra-organ space.
The present microspheres may also be used to fill intra-organ spaces to improve mechanical functioning of organs, particularly the heart and lung. In heart failure, the cardiac chambers may become enlarged to compensate for stress. Dilation of the cardiac chambers adversely affects the pumping function of the heart, resulting in further heart failure. In addition, the enlarged cardiac chamber creates a sac in which stationary blood can form a clot, which can travel to the brain and result in stroke. The same process occurs in the lung; an injured lung can form bullous sacs in a disease known as bullous emphysema. Such bullous sacs are isolated areas for infection and can mechanically compress healthy lung. One remedy for these situations is to use a mechanical bulking agent to fill the intra-organ space (cardiac chamber in the heart, or bullous sac in the lung). In this application, the microspheres are suspended in a biocompatible carrier and are injected into the intra-organ space using a needle or catheter. The microspheres swell upon exposure to physiological fluids and fill the intra-organ space.
In addition voids may be created by surgical procedures such as a biopsy, removal of a tumor, tooth extraction, or removal of infected or damaged tissue. In any of these cases the void may be filled through administering the present microspheres. Filling such voids may reduce the incidence of infection and minimize abnormal appearance of external tissue.
Microspheres, made using the present process, may be used in treatment of wounds, whether surgical (including surgical incision) or accidental (including, for example, cuts, scrapes, abrasions, burns, and sores), to provide treatments such as absorbing fluids, providing padding, and/or delivering medications. For these applications the microspheres may be contained within a sterile covering that is permeable to fluids and/or medications, such as a cotton or synthetic gauze. This microsphere containing item is called a sachet. The microspheres used in this manner may be dry (a powder; not in a suspension), and the rapid and high capacity of swell properties are used for fluid absorption. A contained quantity of microspheres may be applied to a wound or in a surgical incision for this purpose. For example, a type of sachet containing the present microspheres may be inserted in a surgical incision to absorb the released body fluids. An external wound covering, including a bandage, cast, or other covering, may be used to support the dry microspheres for application over a wound to absorb released body fluids. The wound covering includes a support material in contact with the microspheres that may contain the microspheres, such as a porous or mesh material. Alternatively, the microspheres may be attached to the support material.
Additionally, the microspheres may comprise a medication, including a pharmaceutical drug or therapeutic agent, that is released over time as the microspheres come into contact with body fluids. Suitable pharmaceutical drugs and therapeutic agents are well known in the art. An extensive list is given by Kabonov et al. in U.S. Pat. No. 6,696,089, which is incorporated herein by reference (in particular, columns 16 to 18). Examples include, but are not limited to, antibacterial agents, antiviral agents, antifungal agents, anti-cancer agents, vaccines, radiolabels, anti-inflammatories, anti-glaucomic agents, anti-histamine drugs, antiangiogenic factors, local anesthetics, general anesthetic agents, antineoplastic agents, antibodies, vitamins, peptides and peptide analogs, enzymes, anti-allergenic agents, circulatory drugs, anti-tubercular agents, anti-anginal agents, anti-protozoan agents, anti-rheumatic agents, narcotics, cardiac glycoside agents, sedatives, hormones and steroids, and the like. The medication may be loaded into the microspheres using various methods known in the art. For example, the microspheres may be imbibed with the medication by swelling the microspheres in a medium containing the medication and allowing it to soak into the microspheres. The microspheres may then be dried or deswelled by removing water by washing with a dehydrating solvent, as described above. Additionally, the medication may be coated onto the microspheres using methods such as spraying, immersion, and the like. The medication may also be directly incorporated into the microspheres during their preparation by adding the medication to the first solution.
Microspheres containing a pharmaceutical drug or therapeutic agent may be incorporated into a sachet or external covering such as a bandage or cast, and applied to internal or external, surgical or accidental, wounds as described above for non-imbibed microspheres. A bandage containing the microspheres containing a therapeutic agent may be applied to an external tissue that is severely injured such as by a laceration or burn, to rapidly stop blood loss and deliver a high concentration of therapeutic agent directly to the site of injury. For example, a bandage containing microspheres loaded with a growth factor, can promote healing and growth of new tissue (Ulubayram et al. (2001) Biomaterials 22(11):1345-1356). This type of bandage may contain both imbibed and dry microspheres for delivery of medication and absorption. Other types of wound coverings may incorporate microspheres to be used in this manner.
Therapeutic agents imbibed into the microspheres may also be delivered using a transdermal patch containing the microspheres. In many disease states, including infections, cancer, and inflammation, it is desirable to achieve a high concentration of therapeutic agent directly at the diseased site, with controlled release of the therapeutic agent into the surrounding tissue. Targeted drug delivery with controlled release may be achieved by using a transdermal patch that incorporates the present microspheres containing the appropriate therapeutic agent(s).
Alternatively, to reach a target site for drug delivery, the microspheres containing the appropriate pharmaceutical drug or therapeutic agent may be delivered to the site using a needle or catheter (Misirli et al. (2005) Journal of Microencapsulation 22(2):167-178). Microspheres containing a pharmaceutical drug or therapeutic agent may also be used in occlusion, void filling, tissue bulking and other medical treatments.
The present invention is further defined in the following Examples. These Examples are given by way of illustration only, and should not be construed as limiting. From the above discussion and these Examples, one skilled in the art can ascertain the essential characteristics of this invention, and without departing from the spirit and scope thereof, can make various changes and modifications of the invention to adapt it to various uses and conditions.
The meaning of abbreviations used is as follows: “min” means minute(s), “h” means hour(s), “sec” means second(s), “μL” means microliter(s), “mL” means milliliter(s), “L” means liter(s), “nm” means nanometer(s), “mm” means millimeter(s), “cm” means centimeter(s), “cm3” means cubic centimeters, “μm” means micrometer(s) or micron(s), “mM” means millimolar, “M” means molar, “g” means gram(s), “mol” means mole(s), “rpm” means revolutions per minute, “wt %” means percent by weight, “cP” means centipoise, “kGy” is kiloGray(s), “F” means French, “G” means gauge. In the designation d204, d is density, the 4 is the temperature of the standard used to compare the densities, typically water at 4° C., and the 20 is the temperature at which the density of the subject material is measured.
Chemicals and other ingredients were purchased from Aldrich (Milwaukee, Wis.) and used as received, unless otherwise specified. Solvents were purchased from EMD Chemical (Darmstadt, Germany) or Aldrich, as specified below. The VA-044 polymerization initiator was used as received from Wako Pure Chemical Industries, Ltd (Richmond, Va.). All cell culture media were purchased from American Type Culture Collection (ATCC, Manassas, Va.).
Method of Measurement of Microsphere Swell
Swell ratio was determined according to a method described in the following reference: Figuly, Garret D., et. al. Macromolecules 1997, 30, 6174-6184. Into a pre-dried, tared, 150 mL coarse fritted funnel was added approximately 1 g of microspheres. The stem of the funnel was sealed with a rubber stopper. The funnel was placed on a filter flask, and about 150 mL of distilled water at room temperature was added to the funnel and its contents. The contents were stirred, if necessary, to fully disperse the water and microspheres. The contents were left undisturbed for 15 min. The stopper was then removed from the stem of the funnel, and suction was applied for 5 min. The stem and the underside of the funnel were then rinsed with ethanol to remove any remaining water droplets, and suction was then continued for an additional 5 min. Any remaining water droplets were wiped off of the funnel. The funnel and contents were then weighed to determine the weight of water retained by the microspheres. Swell was calculated as follows:
swell=[(total mass of wet microspheres+funnel)−(total mass of dry microspheres+funnel)]/mass of dry microspheres
=[wet mass of microspheres−dry mass of microspheres]/dry mass of microspheres
=mass water retained (g)/mass of dry microspheres (g)
In a 5 L round-bottom, three-necked flask equipped with an overhead stirrer, thermometer, reflux condenser, and nitrogen inlet port was prepared a solution of 36.0 g ethyl cellulose, 1200 mL of chloroform, and 570 g of methylene chloride (solution A). The mixture was stirred at 100 rpm until the ethyl cellulose dissolved; then the agitator was increased in speed to 180 rpm to create a slight vortex. In a second flask, was prepared a solution of 1.50 g methyl cellulose, 3.00 g N,N′-methylenebisacrylamide (2.3 Mol % of monomer), 26.01 g Triton™ X405 (polyoxyethylene (40) isooctylphenyl ether—70% solution in water), and 149.4 g water (solution B). In a third separate flask was mixed 58.5 g of acrylic acid and 81 g of a 25% aqueous sodium hydroxide solution (to reach a pH between 5 and 6) (solution C). This acrylic acid solution was then added to the water solution B.
At this point, while rapidly stirring the mix of Solutions B and C, 0.15 g of the water-soluble azo initiator VA-044 (2,2′-azobis(2-[2-imidazolin-2-yl])propane dihydrochloride) was added, and the resulting solution was stirred for 5 min. This solution (the “first solution”) was then added to the round-bottom flask containing solution A (the “second solution”). The resulting reaction mixture was allowed to stir (the “first suspension”) at 180 rpm for about 1 h at room temperature. The first suspension was then heated to 51° C. and stirred at 180 rpm for an additional 10 h at that temperature to allow substantial microsphere formation (the “second suspension”). The second suspension was then stirred at 180 rpm for another 14 h at room temperature to ensure complete polymerization. After this time, approximately 1200 mL of methanol was slowly added to the second suspension to remove water from the microspheres, and the microspheres were allowed to stir an additional hour. The microspheres were then filtered and washed with an additional 250 mL of methanol. They were filtered again and finally washed with 250 mL of ethanol. They were then dried in a nitrogen purged vacuum oven set at 100° C. The resulting microspheres were white in color. The final yield of dried microspheres was 73.4 g.
The resulting dried microspheres exhibited diameters generally ranging from 25 microns to 250 microns as measured from photos acquired via scanning electron microscopy. Microsphere swell was tested as described in General Methods. When exposed to water, the microspheres absorbed 89 g of water/g of microspheres.
The microspheres were placed on a glass slide that was then placed under a microscope lens. A drop of water was placed on the slide. Movement of the water front across the slide was observed via a high speed multiple exposure digital camera, taking pictures at 2 frames per second. The images were recorded, as well as the time it took for the water front to move across the slide. The microspheres reached nearly maximal size in 4 sec. After 14 sec only a slight increase in microsphere size was observed. Images of the microspheres are shown in
Swelled microspheres were observed under a light microscope. It was observed that microspheres in close contact with each other did not remain spherical, but instead showed deformation of the boundary surfaces thereby increasing the contact area between adjacent microspheres. The extent of the contacts between the swelled and deformed microspheres nearly eliminated open spaces between microspheres, as seen in
Sphericity measurements of microspheres were made on a bulk scale using the Beckman-Coulter™ RapidVUE® particle analysis system (Hialieah, Fla.), using an adaptive threshold value of 56%. A 20 mg sample of microspheres was suspended in 75 mL of water (swelled microspheres), a 50 mg sample of microspheres was suspended in 75 mL of DMSO (unswelled microspheres), and both samples were assayed in the particle analyzer. The results showed that both swelled and unswelled microspheres were close to spherical with a measured sphericity centered near 95%, indicating a high degree of sphericity for the microspheres prepared by the process described in this Example. A picture of unswelled microspheres is shown in
All of the samples for this study were prepared in the following manner, where the only ingredient that was varied was the amount of the crosslinking agent N,N′-methylenebisacrylamide. Values for this ingredient are displayed in Table 1 as both gram quantity of crosslinking agent, and Mol % of crosslinking agent with respect to the sum of the moles of monomer+crosslinking agent in the reaction mixture.
In a 1 L round-bottom, three-necked flask equipped with an overhead stirrer, thermometer, reflux condenser, and nitrogen inlet port was prepared a solution of 12.0 g ethyl cellulose, 400 mL of chloroform, and 190 g of methylene chloride (solution A). The mixture was stirred at 150 rpm until the ethyl cellulose dissolved; then the agitator was increased in speed to 250 rpm to create a slight vortex. In a second flask was prepared a solution of 0.50 g methylcellulose, varying amounts (see Table 1 below) of N,N′-methylenebisacrylamide, 8.67 g Triton™ X-405-70% solution, and 49.8 g water (solution B). In a third separate flask was mixed 19.5 g acrylic acid and 25 g of a 25% aqueous sodium hydroxide solution (to reach a pH of 5.3) (solution C). This acrylic acid solution was then added to the water solution B.
At this point, while rapidly stirring the mixture of solutions B and C, 0.05 g of the water soluble azo initiator VA-044 (2,2′-azobis(2-[2-imidazolin-2-yl])propane dihydrochloride) was added, and the resulting solution was stirred for 5 min. This solution (the “first solution”) was then added to the round-bottom flask containing solution A (the “second solution”). The reaction mixture, which formed a suspension, was allowed to stir at 250 rpm for about 1 h at room temperature (the “first suspension”). The first suspension was then heated to 51° C. and stirred at 250 rpm for an additional 8.5 h at that temperature to allow substantial microsphere formation (the “second suspension”). The second suspension was then stirred at 250 rpm for another 14 h at room temperature to ensure complete polymerization. After this time, approximately 800 mL of methanol was slowly added to the second suspension, to remove water from the microspheres, and the microspheres were allowed to stir an additional hour. The microspheres were then filtered and washed with additional methanol. They were filtered again and finally washed with ethanol. They were then dried in a nitrogen purged vacuum oven set at 100° C. The resulting microspheres were white in color. The microsphere yield for each sample is given in Table 1.
Microsphere swell was tested as described in General Methods. Results of water uptake by each sample of microspheres having a different amount of crosslinking agent, also calculated as mole percent of crosslinker, are shown in Table 1 and a graph of this data is shown in
As can be observed from Table 1 and the graph in
All of the samples for this study were prepared in the following manner, where the only ingredient that was varied was the amount of sodium hydroxide solution added to adjust the pH of the acrylic acid monomer. Values for this ingredient are displayed in Table 2.
In a 1 L round-bottom, three-necked flask equipped with an overhead stirrer, thermometer, reflux condenser, and nitrogen inlet port was prepared a solution of 12.0 g ethyl cellulose, 400 mL chloroform, and 190 g methylene chloride (solution A). The mixture was stirred at 150 rpm until the ethyl cellulose dissolved; then the agitator was increased in speed to 250 rpm to create a slight vortex. In a second flask, was prepared a solution of 0.50 g methyl cellulose, 1.00 g N,N′-methylenebisacrylamide (2.3 Mol %), 8.67 g Triton™ X-405 (70% solution), and 49.8 g water (solution B). In a third separate flask was mixed 19.5 g acrylic acid and varying amounts (see Table 2) of a 25% aqueous sodium hydroxide solution (solution C). The total volume of solution C was made constant for each sample by addition of water. The pH of each sample acrylic acid solution C was measured (Table 2). This acrylic acid solution was then added to the water solution B.
At this point, while rapidly stirring the mixture of Solutions B and C, 0.05 g of the water soluble azo initiator VA-044 (2,2′-azobis(2-[2-imidazolin-2-yl])propane dihydrochloride) was added, and the resulting solution was stirred for 5 min. This solution (the “first solution”) was then added to the round-bottom flask containing solution A (the “second solution”). The reaction mixture was allowed to stir at 250 rpm for about 1 h at room temperature, forming the “first suspension”. The first suspension was then heated to 51° C., and stirred at 250 rpm for an additional 8.5 h at that temperature to allow substantial microsphere formation (the “second suspension”). The second suspension was then stirred at 250 rpm for another 14 h at room temperature to ensure complete polymerization. After this time, approximately 800 mL of methanol was slowly added to the second suspension to remove water from the microspheres, and the microspheres were allowed to stir an additional hour. The microspheres were then filtered and washed with additional methanol. They were filtered again and finally washed with ethanol. They were then dried in a nitrogen purged vacuum oven set at 100° C. The resulting microspheres were white in color. The microsphere yield for each sample is given in Table 2.
The swell of microspheres prepared in each sample was tested as described in General Methods. Results of water uptake by each sample of microspheres having a different pH of solution C are shown in Table 2 and graphed in
As can be observed from the data in Table 2 and
A total of 15 batches of microspheres (1079 g) prepared according to Example 1 were combined and roll milled for 3 h to ensure good mixing. The microspheres were then passed through sieves as follows.
Eight-inch diameter sieves of desired sizes were stacked from the smallest opening at the bottom (nested into a pan) to the largest opening at the top. The sieve sizes used for separating the microspheres were:
The entire sample was sieved (eliminating the need for riffling to obtain a representative sample). For this large sample, a small portion was sieved followed by another small portion. Aliquots were used that covered a sieve to a depth of about 32 mm to 48 mm. A cover was put on the top sieve and the stack of sieves was placed on a Gilson SS-5 Vibratory 3-In. Sieve Shaker. Each sample was run for 10 min with both the vibration and the tap activated.
The weights of seven containers were recorded and each was designated for a specific sieve fraction. Each sieve (or pan) was then dumped into its designated container. A Sigma-Aldrich Zerostat3 gun was used as needed to neutralize the static charge carried by the microspheres, and facilitate emptying each sieve. The sieves were restacked and the process repeated as many times as needed to process the entire sample. After the final portion was sieved, a brush was rubbed across the bottom of the sieve being dumped to dislodge near-sized particles wedged into the sieve openings. The net weight of each container was then determined and is given in Table 3. These results show that the predominance of microspheres ranged in size between 38 and 500 microns, with the 125 to 500 micron size being most prevalent.
The particle size for the sample recovered from each specific sieve was determined as follows. A Beckman Coulter Multisizer 3 was filled with a filtered 2:1 by volume methanol:glycerol mixture into which 3% (by weight) lithium chloride had been dissolved. An aperture tube was chosen which was suitable for the sieve fraction being measured. An aperture tube can detect particles with diameters falling in a range from 2 to 60% of the aperture diameter, and the largest particle dictates the size aperture. A 2000 micron aperture tube was used for the unsieved microspheres and for the >500 fraction. A 200 micron aperture tube was used for the 38-75 micron fraction and for the <38 micron fraction. For the intervening fractions a 1000-micron aperture tube was used. A round-bottomed beaker was filled with about 400 mL of the methanol/glycerol/lithium chloride electrolyte and placed on the sampling platform with the aperture tube immersed in the electrolyte.
A spatula was used to stir and to extract from each sieve sample an aliquot of the microspheres to be measured that was added to a 20 mL cuvette containing methanol. The sample was placed in an ultrasonic bath for ten seconds. A medicine dropper was then used to transfer a portion of this slurry to a beaker sitting on the sampling platform. During the extraction the particles remained in motion so that a representative aliquot could be obtained. This was assured by using the medicine dropper as a stirrer, while at the same time continually drawing in and expelling the slurry. Either the entire content of the medicine dropper was used or, if only a portion was used, the dropper was rapidly moved from cuvette to the beaker and the portion to be used was spurted back into the beaker (this avoids size segregation caused by settling within the dropper). This transfer step was repeated as needed to bring the Multisizer's coincidence indicator to about 5%. The stirrer was then activated and set to a speed as high as possible without the risk of drawing air bubbles into the liquid. A manually controlled run was then begun, and stopped after 60 to 75 sec. The Multisizer data is shown in
Thus the microspheres may be separated into specific sized samples, each containing a predominant size of microsphere, with some variation in size. The size variation is within +/−30% of the median for 90% of the microspheres in the sample for all but the sample B. Most samples have +/−20% variation, and one has +/−16% variation.
The swell capacity was measured for each of samples B through F using the procedure described in the General Methods. Each sample had a maximum swell that was greater than 70 grams of water per gram of microspheres, as shown in Table 5. The samples with larger sizes of microspheres, E and F, had a small increase in swell capacities above those of the smaller microsphere samples, but overall the microsphere size did not greatly affect the swell capacity.
This example compares the properties of hydrogel microspheres prepared using the process described in present Example 1 with those of hydrogel microspheres prepared for comparison using the method of Example 2 of U.S. Pat. No. 6,218,440.
Following the method of Example 2 of U.S. Pat. No. 6,218,440, in a 1 L round-bottom flask equipped with an overhead stirrer, thermometer, reflux condenser, and nitrogen inlet was prepared a solution of 12.0 g ethyl cellulose and 590 g methylene chloride (solution A). The mixture was stirred at 250 rpm until the ethyl cellulose dissolved; and the agitator was then maintained at that speed to create a slight vortex. In a second flask, was prepared a solution of 0.50 g methyl cellulose, 1.00 g N,N′-methylenebisacrylamide, 8.67 g Triton™ X-405 (70% solution), and 49.8 g water (solution B). In a third separate flask was mixed 19.5 g acrylic acid and 19.5 g of a 25% aqueous sodium hydroxide solution (to reach a pH of between 5 and 6) (solution C). This acrylic acid solution was then added to the water solution B.
At this point, while rapidly stirring the mix of Solutions B and C, three separate solutions were prepared as follows: (a) 2.00 g of ammonium persulfate in 3.0 g of water (solution D); (b) 1.2 g of sodium hydrosulfide in 0.2 g of water (solution E); and (c) 1.2 g of Iron III chloride in 0.2 g of water (solution F).
While rapidly stirring the mix of solutions B and C, solution D was added and stirred for 5 min. The combined solution of B, C, and D was then added to the round-bottom flask containing solution A. After this addition, the remaining catalyst solutions E and F were added, and the reaction mixture was allowed to stir for an additional 20 h at room temperature. After this time approximately 350 mL of methanol was added to the reaction mixture, and the microspheres were allowed to stir for an additional hour. The resulting microspheres were then filtered and an additional 400 mL of methanol was then used to wash the microspheres. Again, the microspheres were filtered and washed with 700 mL of ethanol. They were then filtered again and placed in a vacuum oven set at 50° C. with a nitrogen purge. After drying, 34.5 g of a pinkish tan product in the form of microspheres was recovered.
Comparative studies were conducted with the microspheres produced as described in present Example 5 and a sample of microspheres prepared according to present Example 1. The Example 5 process microspheres were pink to tan, while the Example 1 process microspheres were white, as determined by visual inspection (Table 7).
Using the test for swelling in the General Methods, the microspheres prepared by the Example 5 process absorbed 46 g of water per g of microspheres, while the Example 1 process microspheres absorbed 98 g of water per g of microspheres.
Diameter ranges of microspheres in the samples were obtained via visual inspection of scanning electron micrographs set at low magnification in order to observe a high population of microspheres. The smallest and largest microspheres seen in each microsphere population were measured and were used as the top and bottom ranges for the diameter, as given in Table 7. The Example 5 microspheres had diameters ranging between 100 and 475 microns, while the Example 1 process microspheres had diameters between 25 and 250 microns. Note that in this present Example 6, a small sample of present Example 1 process microspheres was assayed, as compared to the sample analyzed in present Example 4, leading to a smaller range in sizes than shown in Table 4.
Bulk density was measured by filling a 50 cm3 tared graduated cylinder with microspheres to the 50 cm3 mark, tapping them down to ensure maximum packing density, and weighing the filled graduated cylinder. Bulk density was then determined by obtaining the weight of the microspheres by difference and dividing that weight by 50 to obtain a density value measured as g/cm3. The Example 1 process microspheres had a much greater density than the Example 5 process microspheres (Table 7).
Fracture of microspheres was tested as follows. A sample of 60 mg of microspheres, prepared as in Example 1 or as in Example 5, was added to 10 mL of either deionized water at pH=2.1 to prevent swell or at pH=7.0 to allow swell. Included in the water was 30 mg of acridine orange, a cationic dye used to stain the microspheres. Each sample of microspheres was filtered out using a micromesh particulate specimen bag, and suspended in 10 mL of solution with the same pH as the starting solution. Each sample was mixed by vortexing, and 2 mL was injected through either a 20 or 21 gauge needle. The injected samples were collected, and the size distribution of the resulting particles was analyzed using the Beckman-Coulter™ RapidVUE® particle analysis system described in Example 2. The injection experiment was repeated 3 times for each sample. As shown in Table 6, both types of unswelled microspheres (in acid solution) passed through 20 gauge and 21 gauge needles without fracture, since the average sample diameters remained constant within the variability of the assay. However, the average diameter of the swelled (in neutral solution) Example 5 type microspheres was greatly reduced after passing through 21 gauge (by about one-third) and 20 gauge (by almost half) needles. In contrast, the average diameter of the swelled (in neutral solution) Example 1 type microspheres was only slightly reduced after passing through 21 gauge and 20 gauge needles. Samples of the Example 5 type microspheres after passage through the 21 gauge needle were viewed by light microscopy. Fragments of microspheres were prevalent.
Microsphere density was measured by the following density gradient technique. A continuous density gradient was established in a vertical column. The gradient was established by partially mixing the column with layers of solvent having different densities. In this case heptane (d204=0.684) and carbon tetrachloride (d2525=1.589) were mixed using different ratios to obtain low and high density mixtures. Then these mixtures were once again mixed through a nitrogen purge system to produce a density blend.
A sample of microspheres was introduced into the gradient, and the microspheres reached an equilibrium point after 24 h, where the density of the liquid equaled the microsphere density. The gradient tubes were temperature controlled at 25° C. Calibrated floats of known densities were used to calibrate the gradient tube in terms of position vs. density. The density of the microspheres was determined using a focusable calibrated magnifying eyepiece to identify the measurable difference between the calibrated float and the sample microspheres, which allowed for a direct conversion to a microsphere density value as given in Table 7. A portion of Example 5 microspheres floated at the top of the gradient indicating a density of less than 0.8 g/cm3, which was not measurable in the system used.
Cross sections of microsphere samples were obtained through standard cutting and microtoming techniques. The microsphere cross sections were examined using standard scanning electron microscopy techniques. As seen in the micrographs of
It can be observed clearly from this comparative example that the present preparative method for hydrogel microspheres described in Example 1 produces more compact, denser, more spherical microspheres, which can swell up to 2× better than microspheres produced using the method of comparative Example 5.
In addition, microspheres prepared by the process of Example 1 are able to deform following swell. Onto a glass microscope slide was placed a group of dry microspheres prepared as in Example 1. The microspheres were covered with a second glass microscope slide and pressure was applied by pressing the top slide against microspheres and the bottom slide by hand, while observing the microspheres under a microscope. When the dried microspheres were pressed in this manner, no noticeable deformation of the microspheres was observed. At this point water was added in sufficient quantity to fully hydrate the microspheres causing them to swell. Pressure was again applied as previously described. When observed under the microscope the microspheres were seen to readily deform under the pressure to a point where it appeared that they were very flat. Upon release of the pressure, the microspheres immediately regained their original spherical shape. Microspheres prepared according to the Example 5 process begin to deform and then fracture when treated with the same pressure.
Individual samples of microspheres prepared as in Example 1 were exposed to either ethanol for 15 min, to 260 nm ultraviolet (UV) light for 30 min, or to gamma radiation at about 28 kGy. In all cases, the resulting sterilized microspheres exhibited similar expansion coefficients to non-sterilized microspheres when exposed to neutral water, indicating that no additional crosslinking or other negative processes had occurred during the sterilization processes.
NIH3T3 human fibroblast cells (obtained from the ATCC, #CRL-1658) were grown in Dulbecco's modified essential medium (DMEM), supplemented with 10% fetal calf serum. A cell culture was challenged with 10 mg of microspheres prepared as in Example 1 according to FDA standard procedure ISO10993-5:1999. The microsphere sample was coated on the bottom of a well in a polystyrene culture plate. The well was then sterilized under UV light and seeded with 50,000-100,000 NIH3T3 cells. The cell culture was incubated for 48 h. The cells grew normally confluent and coated the well bottom, growing up to the edges of the microspheres. This result demonstrated a lack of cytotoxicity of the microspheres.
The testing was done using J774 Macrophage cultures according to FDA standard procedure ISO10993-5:1999. The J774 Macrophage cells were obtained from ATCC (#TIB-67) and were grown in DMEM supplemented with 10% fetal bovine serum.
A J774 mouse peritoneal macrophage cell culture was challenged with 10 mg of microspheres prepared as in Example 1. The microsphere sample was coated on the bottom of a well in a polystyrene culture plate. The well was then sterilized under UV light and seeded with J774 cells. The cell culture was incubated for 48 h. The cell culture was then analyzed for TNF-α (tumor necrosis factor alpha), an indicator of inflammatory response, using an ELISA assay, as described by Lara et al. (Journal of Dental Research 82(6):460-465, 2003). The assay was carried out using a kit purchased from R&D systems (catalog #MTA00), which utilizes a polyclonal antibody against mouse TNF-α. The TNF-αtiter was similar to the negative control (a blank well), which indicated the non-inflammatory nature of the microspheres.
The testing was done using human red blood cells according to FDA standards (ISO10993-4:2002 standards). The red blood cells were obtained from human donors and were diluted to a 5% solution of red blood cells in phosphate buffered saline (1× concentration, pH 7.4).
Dilutions of microspheres prepared as in Example 1 were made in phosphate buffered saline at concentrations of 4.0 μg/mL to 500 μg/mL. The red blood cells were challenged with the microsphere samples by incubating 500 μL of each microsphere solution with 25 μL of 5% red blood cells. The red blood cells with microspheres were incubated for 30 min with agitation. The red blood cells were then analyzed for hemolysis, using spectrophotometry, as described by Malinauskas (Artificial Organs 21(12):1255-1267, 1997). The extent of hemolysis was determined by measuring hemoglobin release from lysed red blood cells. The amount of hemoglobin released by the sample was measured spectrophotometrically at 540 nm. The hydrogel microspheres induced 0.0% hemolysis at a microsphere concentration of 500 μg/mL. This indicated the non-hemolytic nature of the microspheres.
Whole blood was extracted from a rabbit and stored in a 15 mL polypropylene tube (Falcon tube). The microspheres prepared as in Example 1 were suspended in the blood approximately 30 min after it was taken from the rabbit. At this time, some minimal amount of clotting was visible in the blood sample. A 10 mg sample of microspheres was suspended in a 5 mL blood sample for 3 min and then observed under a light microscope. The exposed microspheres exhibited similar expansion coefficients (see Example 13) to microspheres that were exposed to neutral water, demonstrating that the microspheres suspended in whole blood had a similar swelling response to those in pure water. Additionally, the time-course of the swelling was approximately the same; the spheres swelled fully in whole blood in seconds.
Solutions containing different concentrations of DMSO were tested for their effect in maintaining the swellable/deformable microspheres prepared as described in Example 1 in an unswelled state. The solutions containing different amounts of DMSO and water listed in Table 8 were prepared in nine separate 0.5 mL Eppendorf test tubes.
To each test tube was added 5 mg of the swellable/deformable microspheres prepared in Example 1. Each tube was then briefly shaken to submerge all of the microspheres, and they were each left to stand for at least 3 min to allow complete microsphere swelling. At this point samples from each tube were taken and imaged under a microscope at 5× magnification. The level of swell of the microspheres in each tube was qualitatively compared. The comparison showed that solutions containing a high concentration of DMSO (solutions 1-3) effectively suppressed microsphere swell. The solution containing 50% water (solution 4) caused very significant swelling of the microspheres, and solutions containing a higher percentage of water (solutions 5-9) produced about the same level of swell in the microspheres as with 50% water.
Three solutions of differing pH were prepared by adding HCl and/or NaOH to water and quantifying the resulting pH with a pH meter. Solutions of pH 2.0; 7.1, and 10.2 were prepared. At this point individual microspheres prepared as described in Example 1 were isolated on a microscope cover slide in their unswelled state. An image of the dry microsphere was recorded at 20× magnification, and the diameter of the microsphere was measured and recorded. A drop (enough to saturate the microsphere) of one of the aqueous solutions of known pH was then added to the microsphere. The hydrated microsphere was then imaged under a microscope at 5× magnification, and the diameter at the widest point was measured and recorded. The volumetric expansion coefficient was then calculated for the microsphere following hydration using the following equation: volume of hydrated microsphere/volume of dry microsphere=volumetric expansion coefficient. This was calculated as follows:
4/3π(dhyd/2)3/4/3π(ddry/2)3
where dhyd=diameter of the hydrated microsphere and
ddry=diameter of the dry microsphere
Each set of conditions was repeated 5 times with a new microsphere each time. The results, shown in Table 9, demonstrated that in an acidic pH solution, the microspheres underwent almost no swell.
A single dry microsphere from a preparation described in Example 1 was isolated on a microscope slide and imaged at 10× magnification. The diameter was recorded. Approximately 0.25 mL of contrast medium MD-76® (supplied by Tyco; Mansfield, Mass.) was added directly to the slide, suspending the microsphere. A time of 3 min was allowed for microsphere/contrast contact. The microsphere was then imaged again using a microscope at 5× magnification, and the diameter was recorded. The volumetric expansion coefficient of the microsphere was computed as described in Example 13. The suspending and measurement process was then repeated an additional 5 times. The average expansion coefficient of microspheres suspended in MD-76® was 4.9, with a range of between 3.6 and 7.4. Experiments were repeated using neutral water in place of MD-76®. From these experiments an average expansion coefficient of 117.5 was obtained, with a range of between 88 and 146.
The experiment was repeated using Ethiodol® (supplied by SAVAGE Laboratories®; Melville, N.Y.). The expansion coefficients of the microspheres in Ethiodol® were lower than those obtained for microspheres in MD-76® as determined by visual inspection.
Microspheres prepared as described in Example 1 with an average dry diameter of approximately 250 microns were suspended in DMSO by mixing in a 60 mL Falcon Tube for 3 min with a Vortex Touch Mixer (speed 10). Suspensions consisted of 2000 mg of the microspheres in 8 mL (250 mg/mL), or 5 mL (400 mg/mL) of DMSO. An attempt was made to inject 2.5 mL of each suspension through each of various sized catheters using a 10 mL syringe. The following catheters were used: a Cordis Vistabritetip guiding catheter (7F), a Medtronic A VE Z2 guiding catheter (6F), and a Cordis PTA dilation catheter, Opta 5 (5F). The 250 mg/mL suspension readily passed through the Medtronic A VE Z2 guiding catheter (6F) and the Cordis Vistabritetip guiding catheter (7F). However, this suspension did not pass through the Cordis PTA dilation catheter, Opta 5 (5F). The microspheres aggregated around the entry point of the lumen of the 5F catheter. The 400 mg/mL suspension was not able to pass through any of the catheters. This solution could not even maintain homogeneity when injected from the 10 mL syringe with no associated catheter, which indicated an over saturation of microspheres in DMSO, Thus this represents an upper limit of concentration for the microspheres, regardless of catheter dimensions.
The microspheres that were passed through the 6F catheter at 250 mg/mL were collected and observed under a light microscope at 10× magnification. No difference in the level of fragmentation was observed for passed microspheres when compared to controls. Passage of the microspheres through the catheter had no visible adverse effects on the physical integrity of the microspheres.
Two samples of microspheres prepared as described in Example 1 were further prepared as in Example 15. The first sample was prepared using 750 mg of microspheres with an average dry diameter near 250 microns and 5 mL of contrast medium MD-76® (150 mg/mL). The second sample was prepared using 1500 mg of microspheres with an average dry diameter of approximately 100 microns, prepared by sieving as described in Example 4, and 5 mL of contrast medium MD-76® (300 mg/mL). Both suspensions readily passed through a Cordis PTA dilation catheter, Opta 5 (5F). As determined in Example 14, the microspheres had an average expansion coefficient of 4.9 in MD-76®.
Suspensions of microspheres that were made as described in Example 1 were further prepared by adding 200 to 1000 mg of microspheres to 5 mL of DMSO in a 15 mL Falcon tube. The suspensions were made 48 h before administration. The porcine studies were conducted on three separate days using adult male pigs. A 12 mL syringe was used to inject each prepared suspension through a 6F catheter. Prior to each attempted injection, the catheter was angiographically guided to the target location. The injection medium and the targeted vasculature for each experiment are summarized in Table 10.
The targeted vasculature was readily located in all attempted injections. The low viscosity of the delivery media facilitated passage of the microspheres through the catheter. Following the injection of the microspheres, contrast medium was injected to angiographically determine if the target vessel had been occluded (diversion of contrast indicated vessel occlusion). Imaging clearly showed blockage of blood flow distal to the target site in each experiment. Dissection of the renal artery showed an occlusion formed by swelled microspheres at the target site. The microspheres successfully occluded porcine renal vasculature and the porcine right coronary artery.
A linear connected tube system consisting in order of a 60 mL syringe, an 80 cm length tube with an in-line clinical grade pressure transducer (#72-4496; Harvard Apparatus) in the center, and a detachable 1.58 mm internal diameter Tygon® tube (AAB00003 B-44-3 Beverage tubing, #TBT-062B, Small Parts, Inc., Miami Lakes, Fla.) was used to test the durability of occlusions formed by microspheres prepared as described in Example 1. This Tygon® tube has an outside diameter of 4.76 mm and a wall thickness of 1.59 mm. The tube with the in-line transducer was AAB00009 B-44-3 Tygon® tubing, which was connected on one end to the syringe, and on the other end to the AAB00003 B44-3 Tygon® tubing through a step down connector (STCR-09/16, Small Parts, Inc., Miami Lakes, Fla.). The AAB00003Tygon® tube was detached from the system and filled with a 2.5 mL aqueous suspension containing various weights (dry weight) of microspheres (250-500 microns dry diameter) as listed in Table 11. The tube was reattached. The syringe was used to gradually impart internal pressure to the Tygon® tube. The pressure needed to dislodge the microspheres from the Tygon® tubing was recorded as readings from the pressure transducer. The results are given in Table 11.
Microsphere occlusions were able to withstand over 1,000 mm Hg (133 kPa) of pressure. The results of this study demonstrated that the swellable/deformable microspheres are capable of occluding high flow, low resistance systems. This system closely models the most difficult scenario to occlude, arterio-venous malformations due to the relatively large diameter of the Tygon® tube. In most other potential applications vessel diameter is constantly reduced in the target tissue, which virtually guarantees occlusion at some level in the vascular tree.
As a comparison, the durability of occlusions formed by microspheres having almost no swell capacity was tested as described in Example 18. These microspheres were those prepared as Sample 9 in Example 2, having a swell capacity of 1.5 gram of water per gram of microspheres which is considered to be non-swellable. The Tygon® tube was detached from the system and filled with a 2.5 mL aqueous suspension containing various weights (dry weight) of the highly crosslinked microspheres (250-500 microns dry diameter, as listed in Table 12. A syringe was used to gradually impart internal pressure to the Tygon® tube. The pressure needed to dislodge the microspheres from the Tygon® tubing is shown in Table 12 for each sample.
In this example the non-swellable nature of the microspheres does not allow for tight packing of the microspheres and extremely inefficient occlusion is achieved, as determined by the low pressure that is adequate to dislodge the occlusion.
Four different suspensions of the microspheres of Example 1 were prepared as the following samples:
Each suspension was injected through a separate 3F Renegade® Fiber braided microcatheter which has a 533 micron inner diameter (Lot#7704153, Boston Scientific, Natick, Mass.). The tip of each microcatheter was placed into a beaker to monitor for any effluent. For all injections, the syringe was constantly agitated to ensure adequate mixing.
From the sample 1 suspension containing swelled microspheres, some microspheres were able to pass through the Renegade® microcatheter, but the vast majority, greater than 95%, remained within the syringe. The sample 2, 3, and 4 suspensions of limited swell microspheres passed through the microcatheters completely. The sample 5 suspension allowed some passage of microspheres, but the microcatheter became blocked after approximately 3 mL of the suspension was delivered. This experiment demonstrated that amounts of 10 mg, 30 mg, and 60 mg of limited swell microspheres were able to pass through a 3F catheter having an inner diameter of 533 microns. Thus a 3F catheter may be used to deliver an amount of swellable/deformable microspheres that is sufficient to form a highly durable occlusion as tested in Example 18.
Into a 1 L round-bottom, three-necked flask equipped with an overhead stirrer, thermometer, reflux condenser, and nitrogen inlet port was prepared a solution of 12.0 g ethyl cellulose, 400 mL chloroform, and 190 g methylene chloride (solution A). The mixture was stirred at 160 rpm until the ethyl cellulose dissolved; then the agitator was increased in speed to 250 rpm to create a slight vortex. In a second flask, was prepared a solution of 0.50 g methyl cellulose, 1.00 g N,N′-methylenebisacrylamide, 8.67 g Triton X-405 (70% solution), and 49.8 g water (solution B). In a third separate flask was mixed 19.5 g acrylic acid, 4.0 g barium hydroxide, and 16.4 g of a 25% aqueous sodium hydroxide solution (to reach a pH of between 5 and 6), and 10 mL of water (solution C). This acrylic acid solution was then added to the water solution B.
At this point, while rapidly stirring the mix of Solutions B and C, 0.05 g of the water-soluble VA-044 initiator (2,2′-azobis(2-1 [2-imidazolin-2-y])propane dihydrochloride) was added and the resulting solution was stirred for 5 min. This solution (the “first solution”) was then added to the round-bottom flask containing solution A (the “second solution”). The reaction mixture was allowed to stir at 250 rpm for about 1 h at room temperature, forming the “first suspension”. The first suspension was then heated to 51° C. and stirred at 250 rpm for an additional 5.5 h at that temperature, and another 15 h at room temperature, forming the “second suspension”. After this time, approximately 400 mL of methanol was slowly added to the second suspension and the microspheres were allowed to stir an additional hour. The microspheres were then filtered and washed with an additional 250 mL of methanol. They were filtered again and finally washed with 250 mL of ethanol. They were then dried in a nitrogen purged vacuum oven set at 100° C.
The resulting microspheres were white in color. The final yield of dried microspheres was 23.1 g. When exposed to water, the microspheres absorbed 86 g of water/g of microspheres. An X-ray imaging analysis clearly indicated that Barium was contained within the microsphere structure allowing excellent imaging of individual microspheres when dry. As the microspheres swelled, the Barium density was diluted so that imaging became more difficult.
The microspheres of Example 1 were suspended in DMSO at concentrations of 10 mg/mL, 30 mg/mL, and 60 mg/mL. Six milliliters of each suspension was loaded into a syringe and injected through a separate 3F Renegade® Fiber braided microcatheter which has a 533 micron inner diameter (Lot#7704153, Boston Scientific, Natick, Mass.). The tip of each microcatheter was placed into a beaker to monitor for any effluent. For all injections, the syringe was constantly agitated to ensure adequate mixing. Using the 10 mg/mL suspension, microspheres passed through the catheter into the beaker, as observed by adding water to the collection beaker. Some microspheres adhered to the walls and plunger of the syringe, most likely caused by static electricity due to the dryness of the air. Similar results were obtained using the 30 mg/mL suspension, with some microspheres passing through the catheter and some adhering to the syringe. Using the 60 mg/mL suspension, the catheter became occluded after a couple milliliters of DMSO passed through the catheter. A control suspension of 10 mg/mL microspheres in water (fully swelled) occluded the catheter immediately, and the occlusion was visible at the entry point of the catheter.
In a 1 L round-bottom, three-necked flask equipped with an overhead stirrer, thermometer, reflux condenser, and nitrogen inlet port was prepared a solution of 6.0 g of ethyl cellulose, 200 mL of chloroform, and 95 g of methylene chloride (solution A). The mixture was stirred at 180 rpm until the ethyl cellulose dissolved. In a second flask, was prepared a solution of 0.25 g of methyl cellulose, 0.419 g of N,N′-methylenebisacrylamide (2.4 Mol % of monomer), 4.335 g of Triton™ X-405 (70% solution), and 26.22 g of water (solution B). In a third separate flask was mixed 9.75 g of methacrylic acid and 9.06 g of a 25% aqueous sodium hydroxide solution (to reach a pH of between 5 and 6) (solution C). This methacrylic acid solution was then added to solution B.
At this point while rapidly stirring the mixture of Solutions B and C, 0.025 g of the water-soluble azo initiator VA-044 initiator (2,2′-azobis(2-[2-imidazolin2-yl])propane dihydrochloride) was added, and the resulting solution was stirred for 5 min to form the “first solution”. The first solution was then added to the round-bottom flask containing solution A (the “second solution”). The resulting reaction mixture was stirred at 412 rpm for about 1.5 h at room temperature, forming the “first suspension”. The stirring speed was reduced to 225 rpm and the first suspension was heated to 51° C. The first suspension was maintained at the same stirring rate and temperature for almost 6 h to allow for substantial microsphere formation, (i.e., the “second suspension”). The second suspension was then stirred at 225 rpm for another 14 h at room temperature to ensure complete polymerization. After this time, approximately 250 mL of methanol was slowly added to the suspension to remove water from the microspheres, and the microspheres were allowed to stir an additional hour. The microspheres were then filtered, washed with an additional 75 mL of methanol, filtered again, and finally washed with 75 mL of ethanol. The microspheres were then dried in a nitrogen purged vacuum oven set at 100° C. The resulting microspheres were white in color. The final yield of dried microspheres was 7.8 g.
The resulting dried microspheres exhibited diameters generally ranging from 40 microns to 150 microns as measured from photos acquired using scanning electron microscopy. Microsphere swell was tested as described in General Methods. When exposed to water, the microspheres absorbed 98 g of water/g of microspheres.
In a 1 L round-bottom, three-neck flask equipped with an overhead stirrer, thermometer, reflux condenser and nitrogen inlet, was prepared a solution of 12.0 g of ethylcellulose (Aldrich No. 200646), 280 mL of phenylethyl acetate (Aldrich No. 290580), 120 mL of ethyl heptanoate (Aldrich No. 112364) and 190 mL of methylene chloride (EMD No. DX0831-6). The mixture was stirred at a rate of 360 rpm until all of the ethylcellulose dissolved, making solution A. In a second flask, a solution was prepared containing 0.50 g of methylcellulose (Aldrich No. 274429), 49.8 g of water, 1.0 g of N,N methylenebisacrylamide (Aldrich No. 146072) and 8.67 g of Tritron X-405 (70% wt solution in water; Aldrich No. 234737), making solution B. In a third separate flask, 19.5 g of acrylic acid and 22.0 g of 25 wt % aqueous sodium hydroxide solution were mixed. The sodium hydroxide solution was added slowly using a pipette while the acrylic acid was stirred in an ice bath, making solution C.
The acrylic acid solution C was added to the water/methylcellulose solution B while vigorously stirring. Then, 0.025 g of the water soluble VA-044 initiator was added. The solution was stirred for 5 min forming the “first solution”. Then, the first solution was added to the ethylcellulose solution A (“second solution”). The reaction mixture was stirred at room temperature for 1 h at 360 rpm forming the “first suspension”. Then, the suspension was heated to 55° C. for 4 h with stirring at 360 rpm, forming the “second suspension”. After this time the second suspension was stirred at 200 rpm overnight at room temperature. The next day, 400 mL of methanol was added using a dropping funnel and stirring was continued for 1 h at room temperature. Microspheres formed during this process were collected by filtration, washed with methanol, washed several times with ethanol, and then dried for 3 days in a vacuum oven at 100° C. with a slight nitrogen purge. The final yield of dried, white microspheres was 24.84 g.
Microsphere swell was tested as described in General Methods. When exposed to water, the microspheres absorbed 82.8 g of water/g of microspheres.
In a 1 L round-bottom, three-necked flask equipped with an overhead stirrer, thermometer, reflux condenser, and nitrogen inlet port was prepared a solution of 6.0 g of ethyl cellulose, 200 mL of chloroform, and 72 mL of methylene chloride (solution A). The mixture was stirred at 250 rpm until the ethyl cellulose dissolved. In a second flask, was prepared a solution of 0.25 g methyl cellulose, 0.51 g of N,N′-methylenebisacrylamide (2.4 Mol % of monomer), 4.335 g of Triton™ X-405 (70% wt solution in water), and 19.0 g of water (solution B). In a third separate flask was mixed 9.75 g of acrylamide and 14.0 g of water (the observed pH was between 5 and 6) (solution C). This acrylamide solution was then added to the crosslinker solution.
Then, while rapidly stirring the mixture of Solutions B and C, 0.025 g of the water-soluble azo initiator VA-044 (2,2′-azobis(2-[2-imidazolin2-yl])propane dihydrochloride) was added, and the resulting solution was stirred for 5 min, forming the “first solution”. The first solution was then added to the round-bottom flask containing solution A (the “second solution”). The resulting reaction mixture was allowed to stir at 244 rpm for about 1 h at room temperature, forming the “first suspension”. The stirring speed was reduced to 225 rpm and the suspension was heated to 49° C. with continued stirring for almost 6 h to allow for substantial microsphere formation (the “second suspension”). The second suspension was then stirred at 224 rpm for another 14 h at room temperature to ensure complete polymerization. After this time, approximately 250 mL of methanol was slowly added to the suspension to remove water from the microspheres, and the microspheres were stirred an additional hour. The microspheres were then filtered and washed with an additional 175 mL of methanol. They were filtered again, washed twice with 150 mL of ethanol, and dried in a nitrogen purged vacuum oven set at 100° C. The resulting microspheres were white in color. The final yield of dried microspheres was 11.2 g.
The resulting dried microspheres exhibited diameters generally ranging from 10 microns to 170 microns as measured from photos acquired using scanning electron microscopy. Microsphere swell was tested as described in General Methods. When exposed to water, the microspheres absorbed 5 g of water/g of microspheres.
Microspheres were prepared using N-hydroxymethyl acrylamide as monomer using a similar procedure. The resulting microspheres absorbed 6 g of water/g of microspheres.
These results suggest that when acrylamide or N-hydroxymethyl acrylamide are used as single monomers, microspheres with low swell are obtained. However, when these monomers are used as a co-monomer with acrylic acid, high swell microspheres are produced, as shown in Examples 26 and 27.
In a 1 L round-bottom, three-necked flask equipped with an overhead stirrer, thermometer, reflux condenser, and nitrogen inlet port was prepared a solution of 6.0 g of ethyl cellulose, 200 mL of chloroform, and 72 mL of methylene chloride (solution A). The mixture was stirred at 250 rpm until the ethyl cellulose dissolved. In a second flask, was prepared a solution of 0.25 g methyl cellulose, 0.50 g N,N′-methylenebisacrylamide (2.4 Mol % of total amount of monomers), 4.335 g Triton™ X-405 (70% wt solution in water), and 25.3 g of water (solution B). In a third separate flask was mixed 8.775 g of acrylic acid and 9.74 g of a 25% aqueous sodium hydroxide solution (solution C). To this solution 0.975 g of acrylamide was added to generate a co-monomer solution with 90% acrylic acid and 10% acrylamide by weight (the pH was observed to be between 4 and 5). The experiment was repeated using a co-monomer ratio of 70% acrylic acid and 30% acrylic acid. The amount of NaOH and the crosslinker were changed to accommodate the monomer ratios, as shown in Table 13. This monomer solution was then added to the water solution (solution B).
Then while rapidly stirring the mixture of Solutions B and C, 0.025 g of the water-soluble azo initiator VA-044 (2,2′-azobis(2-[2-imidazolin2-yl])propane dihydrochloride) was added, and the resulting solution was stirred for 5 min, forming the “first solution”. The first solution was then added to the round-bottom flask containing solution A (the “second solution”). The resulting reaction mixture was stirred (see Table 13 for stirring rates) for about 1 h at room temperature, forming the “first suspension”. The stirring speed was reduced to about 225 rpm and the suspension was heated to 50.5° C. with continued stirring for almost 6 h to allow for substantial microsphere formation (i.e., the “second suspension”). The second suspension was then stirred at 225 rpm for another 14 h at room temperature to ensure complete polymerization. After this time, approximately 250 mL of methanol was slowly added to the suspension to remove water from the microspheres, and the microspheres were stirred for an additional hour. The microspheres were then filtered and washed with an additional 75 mL of methanol, filtered again, washed twice with 75 mL of ethanol and dried in a nitrogen purged vacuum oven set at 100° C. The resulting microspheres were white in color.
The diameter of the resulting dried microspheres was measured from photos acquired using scanning electron microscopy and the swell was determined as described in General Methods. The results are summarized in Table 14. In contrast to microspheres prepared using acrylamide alone (Example 25), microspheres prepared using acrylic acid and acrylamide as co-monomers had high swell.
In three separate 1 L round-bottom, three-necked flasks, each equipped with an overhead stirrer, thermometer, reflux condenser, and nitrogen inlet port, solutions of 6.0 g of ethyl cellulose, 200 mL of chloroform, and 72 mL of methylene chloride (solution A) were prepared. Each mixture was stirred at 250 rpm until the ethyl cellulose dissolved. In a series of second flasks, three solutions of 0.25 g methyl cellulose, varying amounts of N,N′-methylenebisacrylamide to give 2.4 Mol % of total amount of monomers (as listed in Table 15), 4.335 g Triton™ X405 (70% wt solution in water), and 25.3 g of water (solution B) were prepared. In a third series of separate flasks, varying amounts of acrylic acid and 25% aqueous sodium hydroxide solution (to reach a pH of between 5 and 6) (solution C) as given in Table 15 were mixed. To these solutions different amounts of 2-hyrdoxyethyl methacrylate (HEMA) were added to generate the co-monomer solutions with acrylic acid and 2-hydroxyethyl methacrylate compositions as given in Table 15. The amount of NaOH and the crosslinker were changed to accommodate the monomer ratios. Each monomer solution was then added to the appropriate solution B.
Then while rapidly stirring the mixtures of Solutions B and C, 0.025 g of the water-soluble azo initiator VA-044 (2,2′-azobis(2-[2-imidazolin2-yl])propane dihydrochloride) was added, and the resulting solutions were stirred for 5 min, forming the “first solutions”. The first solutions were then added to the round-bottom flasks containing the appropriate solution A (the “second solutions”). The resulting reaction mixtures were stirred (see Table 15 for stirring rates) for about 1 h at room temperature, forming the “first suspensions”. The stirring speed was reduced to about 225 rpm and the suspensions were heated to 51° C. with continued stirring for almost 6 h to allow for substantial microsphere formation (the “second suspensions”). The suspensions were then stirred for another 14 h at room temperature to ensure complete polymerization. After this time, approximately 250 mL of methanol was slowly added to the suspensions to remove water from the microspheres, and the microspheres were stirred for an additional hour. The microspheres were then filtered, washed with an additional 75 mL of methanol, filtered again, washed twice with 75 mL of ethanol, and dried in a nitrogen purged vacuum oven set at 100° C. The resulting microspheres were white in color.
The diameter of the resulting dried microspheres was measured from photos acquired using scanning electron microscopy and the swell was determined as described in General Methods. The results are summarized in Table 16.
In three separate 1 L round-bottom, three-necked flasks, each equipped with an overhead stirrer, thermometer, reflux condenser, and nitrogen inlet port, solutions of 6.0 g of ethyl cellulose, 200 mL of chloroform, and 72 mL of methylene chloride (solution A) were prepared. Each mixture was stirred at 250 rpm until the ethyl cellulose dissolved. In a series of second flasks, three solutions of 0.25 g methyl cellulose, varying amounts of N,N′-methylenebisacrylamide to give 2.4 Mol % of total amount of monomers (as listed in Table 17), 4.335 g Triton™ X-405 (70% wt solution in water), and 25.3 g of water (solution B) were prepared. In a third series of separate flasks, varying amounts of acrylic acid and 25% aqueous sodium hydroxide solution (to reach a pH of between 5 and 6) (solution C) as given in Table 17 were mixed. To these solutions different amounts of 2-hyrdoxyethyl acrylate (HEA) were added to generate the co-monomer solutions with acrylic acid and 2-hydroxyethyl methacrylate compositions as given in Table 17. The amount of NaOH and the crosslinker were changed to accommodate the monomer ratios. Each monomer solution was then added to the appropriate solution B.
Then while rapidly stirring the mixtures of Solutions B and C, 0.025 g of the water-soluble azo initiator VA-044 (2,2′-azobis(2-[2-imidazolin2-yl])propane dihydrochloride) was added, and the resulting solutions were stirred for 5 min, forming the “first solutions”. The first solutions were then added to the round-bottom flasks containing the appropriate solution A (the “second solutions”). The resulting reaction mixtures were stirred (see Table 17 for stirring rates) for about 1 h at room temperature, forming the “first suspensions”. The stirring speed was reduced to about 225 rpm and the suspensions were heated to 51° C. with continued stirring for almost 6 h to allow for substantial microsphere formation (the “second suspensions”). The suspensions were then stirred for another 14 h at room temperature to ensure complete polymerization. After this time, approximately 250 mL of methanol was slowly added to the suspensions to remove water from the microspheres, and the microspheres were stirred for an additional hour. The microspheres were then filtered, washed with an additional 75 mL of methanol, filtered again, washed twice with 75 mL of ethanol, and dried in a nitrogen purged vacuum oven set at 100° C. The resulting microspheres were white in color.
The diameter of the resulting dried microspheres was measured from photos acquired using scanning electron microscopy and the swell was determined as described in General Methods. The results are summarized in Table 18.
In a 5 L round-bottom, three-necked flask equipped with an overhead stirrer, thermometer, reflux condenser, and nitrogen inlet port was prepared a solution of 36.0 g ethyl cellulose, 1200 mL of chloroform, and 570 g of methylene chloride (solution A). The mixture was stirred at 100 rpm until the ethyl cellulose dissolved; then the agitator was increased in speed to 200 rpm to create a slight vortex. In a second flask, was prepared a solution of 1.50 g methyl cellulose, 3.00 g N,N′methylenebisacrylamide (2.3 Mol % of monomer), 26.01 g of Triton™ X-405 (polyoxyethylene (40) isooctylphenyl ether—70% solution in water), and 149.4 g of water (solution B). In a third separate flask was mixed 58.5 g of acrylic acid and 75 g of a 25% aqueous sodium hydroxide solution (to reach a pH between 5 and 6) (solution C). This acrylic acid solution was then added to the water solution B.
At this point, while rapidly stirring the mixture of Solutions B and C, 0.15 g of the water-soluble azo initiator VA-044 (2,2′-azobis(2-[2-imidazolin-2-yl])propane dihydrochloride) was added, and the resulting solution was stirred for 5 min. This solution (the “first solution”) was then added to the round-bottom flask containing solution A (the “second solution”). The resulting reaction mixture was allowed to stir (the “first suspension”) at 200 rpm for about 1 h at room temperature. The first suspension was then heated to 51° C. and stirred at 140 rpm for an additional 10 h at that temperature to allow substantial microsphere formation (the “second suspension”). The second suspension was then stirred at 140 rpm for another 14 h at room temperature to ensure complete polymerization. After this time, approximately 1200 mL of methanol was slowly added to the second suspension to remove water from the microspheres, and the microspheres were allowed to stir for an additional hour. The microspheres were then filtered, washed with an additional 250 mL of methanol, filtered again, and finally washed with 250 mL of ethanol. A portion of the microspheres was then dried in a nitrogen purged vacuum oven set at room temperature for 144 h. The resulting microspheres were white in color. Microsphere swell was tested as described in General Methods. When exposed to water, the microspheres absorbed 120 g of water/g of microspheres.
For comparison, a second batch of microspheres produced from the procedure was dried in a nitrogen purged vacuum oven set at 100° C. for 52 h. The resulting white microspheres were tested for swell as described in General Methods. When exposed to water, the microspheres absorbed 90 g of water/g of microspheres.
These results indicate that a slow room temperature drying process leads to microspheres that exhibit a greater degree of swell than those prepared using a heated drying process.
Preparation of highly swellable microspheres can be attained through the use of a highly hydrophilic monomer, such as sodium acrylate, low crosslink density, and a room temperature drying process. The following process illustrates such a preparation.
In a 5 L round-bottom, three-necked flask equipped with an overhead stirrer, thermometer, reflux condenser, and nitrogen inlet port was prepared a solution of 36.0 g ethyl cellulose, 1200 mL of chloroform, and 570 g of methylene chloride (solution A). The mixture was stirred at 100 rpm until the ethyl cellulose dissolved; then the agitator was increased in speed to 200 rpm to create a slight vortex. In a second flask, was prepared a solution of 1.50 g methyl cellulose, 0.10 g N,N′-methylenebisacrylamide (0.08 Mol % of monomer), 26.01 g Triton™ X-405 (polyoxyethylene (40) isooctylphenyl ether—70% solution in water), and 96.9 g water (solution B). In a third separate flask was mixed 58.5 g of acrylic acid and 127.5 g of a 25% aqueous sodium hydroxide solution (to reach a pH between 9 and 10) (solution C). This acrylic acid solution was then added to the water solution B.
At this point, while rapidly stirring the mixture of Solutions B and C, 0.15 g of the water-soluble azo initiator VA-044 (2,2′-azobis(2-[2-imidazolin-2-yl])propane dihydrochloride) was added, and the resulting solution was stirred for 5 min. This solution (the “first solution”) was then added to the round-bottom flask containing solution A (the “second solution”). The resulting reaction mixture was allowed to stir (the “first suspension”) at 200 rpm for about 1 h at room temperature. The first suspension was then heated to 51° C. and stirred at 140 rpm for an additional 10 h at that temperature to allow substantial microsphere formation (the “second suspension”). The second suspension was then stirred at 140 rpm for another 14 h at room temperature to ensure complete polymerization. After this time, approximately 1200 mL of methanol was slowly added to the second suspension to remove water from the microspheres, and the microspheres were allowed to stir for an additional hour. The microspheres were then filtered, washed with an additional 250 mL of methanol, filtered again, and finally washed with 250 mL of ethanol. A portion of the microspheres were then dried in a nitrogen purged vacuum oven set at room temperature for 64 h. The resulting microspheres were white in color. Microsphere swell was tested as described in General Methods. When exposed to water, the microspheres absorbed 269 g of water/g of microspheres. The average bulk density of these microspheres was measured to be 0.884±0.061 g/cm3 (average and standard deviation of 5 determinations).
For comparison, a second batch of microspheres produced from the procedure was dried in a nitrogen purged vacuum oven set at 100° C. for 64 h. The resulting white microspheres were tested for swell as described in General Methods. When exposed to water, the microspheres absorbed 120 g of water/g of microspheres.
Total yield of microspheres (dried under both conditions) was 81.6 g. A scanning electron micrograph examination of the product beads indicated spherical size ranges from 50 microns to 250 microns.
In a 1 L round-bottom, three-necked flask equipped with an overhead stirrer, thermometer, reflux condenser, and nitrogen inlet port was prepared a solution of 6.0 g of ethyl cellulose, 269 mL of chloroform, and 97 g of methylene chloride (solution A). The mixture was stirred at 244 rpm until the ethyl cellulose dissolved. In a second flask, was prepared a solution of 0.25 g methyl cellulose, 0.175 g of N,N′-methylenebisacrylamide (2.4 Mol % of monomer), 4.335 g Triton™ X-405 (polyoxyethylene (40) isooctylphenyl ether—70% solution in water), and 5.0 g of water (solution B). In a third separate flask was mixed 9.75 g of 4-styrenesulfonic acid, sodium salt hydrate (0.047 mol) and 17.24 g of a 10% HCl solution (0.047 mol; to convert the sodium salt of the monomer to the acid form), also 28.9 g of water was added to this solution (to reach a pH of 0) (solution C). The monomer solution was then added to the crosslinker solution (solution B). The total amount of water in the medium was 49.4 g, including that from the HCl. The amount of chloroform and methylene chloride were increased relative to the amounts used with acrylic acid (Example 1) in order to maintain a similar ratio of organic to water solutions.
At this point while rapidly stirring the mixture of solutions B and C, 0.025 g of the water-soluble azo initiator VA-044 (2,2′-azobis(2-[2-imidazolin2-yl])propane dihydrochloride) was added, and the resulting solution was stirred for 5 min. This solution (the “first solution”) was then added to the round-bottom flask containing solution A (the “second solution”). The resulting reaction mixture was allowed to stir (the “first suspension”) at 235 rpm for about 1 h at room temperature. The stirring speed was reduced to 224 rpm and the first suspension was heated to 50.3° C. The suspension was maintained at the same stirring rate and temperature for almost 6 h to allow for substantial microsphere formation (the “second suspension”). The second suspension was then stirred at 223 rpm for another 14 h at room temperature to ensure complete polymerization. After this time, approximately 250 mL of methanol was slowly added to the second suspension to remove water from the microspheres, and the microspheres were stirred an additional hour. The microspheres were then filtered, the material obtained was too soft to be isolated. The soft mass was washed with acetone and then filtered again. The material was further washed with 100 mL of methanol and washed again twice with 80 mL portions of ethanol. Finally the solids were dried in a nitrogen purged vacuum oven set at 100° C. The resulting microspheres where obtained as a fine powder with a yellow tint. The final yield of dried microspheres was 5.26 g.
The resulting dried microspheres exhibited diameters generally ranging from 10 microns to 70 microns as measured from photos acquired via scanning electron microscopy. Microsphere swell was tested as described in General Methods. When exposed to water, the microspheres absorbed 471 g of water/g of microspheres.
In a 1 L round-bottom, three-necked flask equipped with an overhead stirrer, thermometer, reflux condenser, and nitrogen inlet port was prepared a solution of 6.0 g of ethyl cellulose, 274 g of chloroform, and 99 mL of methylene chloride (solution A). The mixture was stirred at 244 rpm until the ethyl cellulose dissolved. In a second flask, was prepared a solution of 0.25 g methyl cellulose, 0.175 g of N,N′-methylenebisacrylamide (2.4 Mol % of monomer), 4.335 g Triton™ X405 (polyoxyethylene (40) isooctylphenyl ether—70% solution in water), and 5.0 g of water (solution B). In a third separate flask was mixed 9.75 g of 4-styrenesulfonic acid, sodium salt hydrate (0.047 mol) and 8.62 g of a 10% HCl solution (0.0236 mol; to convert 50% of the sodium salt of the monomer to the acid form), also 32.3 g of water was added to this solution (to reach a pH of 0) (solution C). The monomer solution was then added to the crosslinker solution (solution B). The total amount of water in the medium was 45.06 g, including that from the HCl. The amount of chloroform and methylene chloride were increased relative to the amounts used with acrylic acid (Example 1) in order to maintain a similar ratio of organic to water solutions.
At this point while rapidly stirring the mixture of solutions B and C, 0.025 g of the water-soluble azo initiator VA-044 (2,2′-azobis(2-[2-imidazolin2-yl])propane dihydrochloride) was added, and the resulting solution was stirred for 5 min. This solution (the “first solution”) was then added to the round-bottom flask containing solution A (the “second solution”). The resulting reaction mixture was allowed to stir (the “first suspension”) at 235 rpm for about 1 h at room temperature. The stirring speed was reduced to 223 rpm and the first suspension was heated to 50.4° C. The suspension was maintained at the same stirring rate and temperature for almost 6 h to allow for substantial microsphere formation (the “second suspension”). The second suspension was then stirred at 223 rpm for another 14 h at room temperature to ensure complete polymerization. After this time, approximately 250 mL of methanol was slowly added to the second suspension to remove water from the microspheres, and the microspheres were stirred an additional hour. The microspheres were then filtered, the material obtained was too soft to be isolated. The soft mass was washed with acetone and then filtered again. The material was further washed with 100 mL of methanol and washed again twice with 80 mL portions of ethanol. Finally the solids were dried in a nitrogen purged vacuum oven set at 100° C. The resulting microspheres where obtained as a fine powder with a yellow tint. The final yield of dried microspheres was 6.29 g.
The resulting dried microspheres exhibited diameters generally ranging from 30 microns to 230 microns as measured from photos acquired via scanning electron microscopy. Microsphere swell was tested as described in General Methods. When exposed to water, the microspheres absorbed 536 g of water/g of microspheres.
In a 1 L round-bottom, three-necked flask equipped with an overhead stirrer, thermometer, reflux condenser, and nitrogen inlet port was prepared a solution of 6.0 g of ethyl cellulose, 200 mL of chloroform, and 72 mL of methylene chloride (solution A). The mixture was stirred at 244 rpm until the ethyl cellulose dissolved. In a second flask, was prepared a solution of 0.25 g methyl cellulose, 0.175 g of N,N′-methylenebisacrylamide (2.4 Mol % of total monomer content: styrene sulfonic acid sodium salt hydrate and acrylic acid), 4.335 g Triton™ X-405 (polyoxyethylene (40) isooctylphenyl ether—70% solution in water), and 17.76 g of water (solution B). In a third separate flask was mixed 4.785 g of 4-styrenesulfonic acid, sodium salt hydrate (0.0236 mol), and 4.785 g of acrylic acid (0.068 mol), also 15.24 g of water was added to this solution (the solution had a pH of 1) (solution C). The monomer solution was then added to the crosslinker solution (solution B).
At this point while rapidly stirring the mix of solutions B and C, 0.025 g of the water-soluble azo initiator VA-044 (2,2′-azobis(2-[2-imidazolin2-yl])propane dihydrochloride) was added, and the resulting solution was stirred for 5 min. This solution (the “first solution”) was then added to the round-bottom flask containing solution A (the “second solution”). The resulting reaction mixture was allowed to stir (the “first suspension”) at 275 rpm for about 1 h at room temperature. The stirring speed was reduced to 222 rpm and the first suspension was heated to 50.4° C. The suspension was maintained at the same stirring rate and temperature for almost 6 h to allow for substantial microsphere formation (the “second suspension”). The second suspension was then stirred at 223 rpm for another 14 h at room temperature to ensure complete polymerization. After this time, approximately 250 mL of methanol was slowly added to the second suspension to remove water from the microspheres, and the microspheres were allowed to stir an additional hour. The microspheres were then filtered, the material obtained was too soft to be isolated. The soft mass was washed with acetone and then filtered again. The material was further washed with 100 mL of methanol and washed again twice with 80 mL portions of ethanol. Finally the solids were dried in a nitrogen purged vacuum oven set at 70° C. over a period of three days. The resulting microspheres where obtained as a fine powder with a yellow tint. The final yield of dried microspheres was 3.65 g.
The resulting dried microspheres exhibited diameters generally ranging from 10 microns to 95 microns as measured from photos acquired via scanning electron microscopy. Microsphere swell was tested as described in General Methods. When exposed to water, the microspheres absorbed 332 g of water/g of microspheres.
In a 1 L round-bottom three-necked flask equipped with an overhead stirrer, thermometer, reflux condenser, and nitrogen inlet port was prepared a solution of 6.0 g of ethyl cellulose, 200 mL of chloroform, and 72 mL of methylene chloride (solution A). The mixture was stirred at 180 rpm until the ethyl cellulose dissolved. In a second flask, was prepared a solution of 0.25 g methyl cellulose, 0.838 g of poly(ethyleneglycol) diacrylate (2.4 Mol % of monomer), 4.335 g Triton™ X-405 (polyoxyethylene (40) isooctylphenyl ether—70% solution in water), and 24.9 g of water (solution B). In a third separate flask was mixed 9.75 g of acrylic acid and 10.82 g of a 25% aqueous sodium hydroxide solution (to reach a pH of between 5 and 6) (solution C). This acrylic acid solution was then added to the water solution (solution B).
At this point while rapidly stirring the mixture of solutions B and C, 0.025 g of the water-soluble azo initiator VA-044 (2,2′-azobis(2-[2-imidazolin2-yl])propane dihydrochloride) was added, and the resulting solution was stirred for 5 min. This solution (the “first solution”) was then added to the round-bottom flask containing solution A (the “second solution”). The resulting reaction mixture was allowed to stir (the “first suspension”) at 327 rpm for about 1 h at room temperature. The stirring speed was reduced to 224 rpm and the first suspension was heated to 50.4° C. The suspension was maintained at the same stirring rate and temperature for almost 6 h to allow for substantial microsphere formation (the “second suspension”). The second suspension was then stirred at 225 rpm for another 14 h at room temperature to ensure complete polymerization. After this time, approximately 250 mL of methanol was slowly added to the second suspension to remove water from the microspheres, and the microspheres were stirred for an additional hour.
The resulting microspheres clumped together at the bottom of the reaction flask into a big mass which was difficult to separate into individual microspheres. A small portion of the collected microsphere mass was removed using tweezers. Various solvents including acetone, methanol, ethanol and hexane were utilized to test if the portion could be separated into individual microspheres. The individual microspheres were observed under a desk optical microscope. Eventually, it was found that water was able to dissolve the beads.
The use of the very hydrophilic crosslinker poly(ethyleneglycol) diacrylate with acrylic acid produced microspheres that were difficult to isolate. It is believed that this crosslinker and similar hydrophilic crosslinkers would work better with more hydrophobic monomers that would provide a better balance of hydrophobic and hydrophilic properties for the microspheres.
Microspheres are made as described in Example 1, and a microsphere suspension is prepared by adding 200 to 1000 mg of microspheres to 5 mL of DMSO in a 15 mL Falcon tube. The suspensions are made 48 h before administration. A 12 mL syringe is used to inject each prepared suspension through a 14F catheter. An endoscope is introduced nasally and is guided down to the level of the gastro-esophageal junction. The catheter is introduced through the endoscope and the suspension is injected into the walls of the lower esophageal sphincter. The injection is made along the muscle layer or deep submucosal layer of the gastric cardia. Multiple injections are performed in a circumferential manner around the esophagus under endoscopic control. Upon contact with physiological fluids, the microspheres swell and become secured in the lower esophageal sphincter.
Microspheres are made as described in Example 1, and a microsphere suspension is prepared by adding 200 to 1000 mg of microspheres to 5 mL of DMSO in a 15 mL Falcon tube. The suspensions are made 48 h before administration. A 12 mL syringe is used to inject each prepared suspension through a 14F catheter. The catheter is introduced into the urethra and the suspension is injected into the walls of the bladder sphincter. Upon contact with physiological fluids, the microspheres swell and become secured in the bladder sphincter.
Microspheres are made as described in Example 1, and a microsphere suspension is prepared by adding 200 to 1000 mg of microspheres to 5 mL of DMSO in a 15 mL Falcon tube. The suspensions are made 48 h before administration. A 12 mL syringe is used to inject each prepared suspension through a 9F catheter. The catheter is introduced percutaneously, via a transrenal approach; the catheter location is visualized by injecting standard contrast medium. The suspension is then injected into the distal ureter. Upon contact with physiological fluids, the microspheres swell and become secured in the distal ureter, resulting in complete occlusion of the ureter. Occlusion is confirmed by injecting standard contrast medium into the catheter.
Microspheres are made as described in Example 1, and a microsphere suspension is prepared by adding 200 to 1000 mg of microspheres to 5 mL of DMSO in a 15 mL Falcon tube. The suspensions are made 48 h before administration. The suspensions are then injected into the skin through a needle of 30 gauge or smaller, using a 10 mL syringe, at the desired site of dermal augmentation. The suspension is injected into the desired skin layer (epidermis, dermis, fat, or subcutaneous layer). Upon contact with physiological fluids, the microspheres swell and become secured at the position of the injection.
Microspheres are made as described in Example 1, and a microsphere suspension is prepared by adding 200 to 1000 mg of microspheres to 5 mL of DMSO in a 15 mL Falcon tube. The suspensions are made 48 h before administration. A 12 mL syringe is used to inject each prepared suspension through a 14F catheter. A colonoscope is introduced rectally and is guided to the site of the diverticular pouch. The catheter is introduced and is placed at the site of the diverticular pouch under colonoscopic guidance. The suspension is injected into the diverticular pouch. Upon contact with physiological fluids, the microspheres swell and fill the diverticular pouch. The microspheres become secured in the diverticulum. Complete filling of the diverticulum is confirmed by barium imaging of the gastrointestinal tract.
Microspheres are made as described in Example 1, and a microsphere suspension is prepared by adding 200 to 1000 mg of microspheres to 5 mL of DMSO in a 15 mL Falcon tube. The suspensions are made 48 h before administration. A 12 mL syringe is used to inject each prepared suspension through a 5F catheter. An endoscope is introduced nasally and is guided down to the pancreatic duct. The catheter is introduced and is placed at the site of the pancreatic duct; the catheter location is visualized by injecting standard contrast medium. The suspension is injected into the pancreatic duct. Upon contact with physiological fluids, the microspheres swell and fill the duct. The microspheres become secured in the pancreatic duct. Complete filling of the pancreatic duct is confirmed by injecting standard contrast medium.
Microspheres are made as described in Example 1, and a microsphere suspension is prepared by adding 200 to 1000 mg of microspheres to 5 mL of DMSO in a 15 mL Falcon tube. The suspensions are made 48 h before administration. A 12 mL syringe is used to inject each prepared suspension through a 5F curved catheter. The catheter is introduced vaginally into the uterus and is placed in the fallopian tube; the catheter location is visualized by injecting standard contrast medium. The suspension is then injected into the fallopian tube. Upon contact with physiological fluids, the microspheres swell and become secured in the fallopian tube, resulting in complete occlusion of the fallopian tube. Occlusion is confirmed by injecting standard contrast medium into the catheter.
Microspheres are made as described in Example 1, and a microsphere suspension is prepared by adding 200 to 1000 mg of microspheres to 5 mL of DMSO in a 15 mL Falcon tube. The suspensions are made 48 h before administration. A 12 mL syringe is used to inject each prepared suspension through a 30 gauge lymphangiogram needle. The needle is introduced trans-scrotally and is placed in the vas deferens; the needle location is visualized by injecting standard contrast medium. The suspension is then injected into the vas deferens. Upon contact with physiological fluids, the microspheres swell and become secured in the vas deferens, resulting in complete occlusion of the vas deferens. Occlusion is confirmed by injecting standard contrast medium into the needle.
Microspheres are made as described in Example 1, and a microsphere suspension is prepared by adding 200 to 1000 mg of microspheres to 5 mL of DMSO in a 15 mL Falcon tube. The suspensions are made 48 h before administration. A 12 mL syringe is used to inject each prepared suspension through a 25 gauge needle. The lower eyelid is pulled down to expose the lower punctum, and the needle is inserted into the punctal opening. The suspension is then injected into the punctum. Upon contact with physiological fluids, the microspheres swell and become secured in the punctum, resulting in complete occlusion of the punctum. Occlusion is confirmed by the increase in tear retention in the treated eye.
Microspheres are made as described in Example 1, and a microsphere suspension is prepared by adding 200 to 1000 mg of microspheres to 5 mL of DMSO in a 15 mL Falcon tube. The suspensions are made 48 h before administration. A 12 mL syringe is used to inject each prepared suspension through a 3F catheter. The catheter is introduced percutaneously and is placed in the orifice of the bronchial artery to be occluded; the catheter location is visualized by injecting standard contrast medium. The suspension is then injected into the bronchial artery. Upon contact with physiological fluids, the microspheres swell, resulting in complete occlusion of the bronchial artery. Occlusion is confirmed by injecting standard contrast medium into the catheter.
Microspheres are made as described in Example 1, and a microsphere suspension is prepared by adding 200 to 1000 mg of microspheres to 5 mL of DMSO in a 15 mL Falcon tube. The suspensions are made 48 h before administration. The suspensions are then injected percutaneously through a needle of 30 gauge or smaller, using a 10 mL syringe, at the desired site of space filling. The suspension is injected into the desired extra-organ space (peritoneum, pericardium, pleura). Upon contact with physiological fluids, the microspheres swell and become secured at the position of the injection.
Microspheres are made as described in Example 1, and a microsphere suspension is prepared by adding 200 to 1000 mg of microspheres to 5 mL of DMSO in a 15 mL Falcon tube. The suspensions are made 48 h before administration. A 12 mL syringe is used to inject each prepared suspension through a 6F catheter. The catheter is introduced percutaneously and is placed in the cardiac chamber to be filled (right atrium, right ventricle, left atrium, or left ventricle). The catheter location is visualized by injecting standard contrast medium. Upon contact with physiological fluids, the microspheres swell and fill the intra-organ space at the position of injection.
Microspheres are made as described in Example 1. A therapeutic agent in an aqueous solution or suspension is added to the microspheres and the microspheres are allowed to imbibe the therapeutic agent. The microspheres are dried and further prepared by adding 200 to 1000 mg of microspheres to 5 mL of DMSO in a 15 mL Falcon tube. The suspensions are made 48 h before administration. The suspensions are then injected percutaneously through a needle of 30 gauge or smaller, using a 10 mL syringe, at the desired site of drug delivery. The suspension is injected into the desired space (skin, peritoneum, pericardium, pleura). Upon contact with physiological fluids, the microspheres swell and become secured at the position of the injection. The drug is released in a controlled fashion at the site of injection.
Microspheres are made as described in Example 1. A therapeutic agent in an aqueous solution or suspension is added to the microspheres and the microspheres are allowed to imbibe the therapeutic agent. The microspheres are dried and further prepared by incorporating the microspheres into a transdermal patch. The patch is applied to the skin at the desired site of drug delivery. Upon contact with physiological fluids, the microspheres swell and the drug is released in a controlled fashion at the site of patch application.
Microspheres are made as described in Example 1. A therapeutic agent in an aqueous solution or suspension is added to the microspheres and the microspheres are allowed to imbibe the therapeutic agent. The microspheres are dried and further prepared by incorporating the microspheres into a bandage. The bandage is applied to the skin at the site of injury and bleeding. Upon contact with physiological fluids, the microspheres swell to stop bleeding, and the drug is released in a controlled fashion at the site of bandage application.
Microspheres are made as described in Example 1. The microspheres are further prepared by incorporating the microspheres into a bandage. The bandage is applied to the skin at the site of injury and bleeding. Upon contact with physiological fluids, the microspheres swell to stop bleeding at the site of bandage application.
This application claims priority under 35 U.S.C. §119 from U.S. Provisional Application Ser. No. 60/791,188, filed Apr. 11, 2006.
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