Colour-coded and sized loadable polymeric particles for therapeutic and/or diagnostic applications and colour-coded syringe comprising the same

ES 2 564 151 T3

DESCRIPTION

Color-coded, chargeable polymeric particles sized for therapeutic and / or diagnostic applications and methods of preparation and use thereof

Background of the invention

Small particles, including microspheres and nanospheres, have many medical uses in diagnostic and therapeutic procedures. In selected clinical applications, it may be advantageous to provide specific sizes of such microspheres and nanospheres to a user. Such sizing of microspheres and nanospheres can allow selective embolization of certain sized blood vessels in specific clinical applications. In addition, it may be advantageous to provide a user with color-coded microspheres or nanospheres to allow easy identification of sized particles in use. Such color-coded microspheres or nanospheres can further be provided in coded delivery or containment devices to enhance user identification and provide visual confirmation of the use of a specifically desired size of microspheres or nanospheres.

Most of the prior art particles used in medical applications are characterized by numerous disadvantages including irritation of the tissues with which they come in contact and the initiation of adverse immune reactions. Additionally, many of the materials used to prepare the prior art particles can degrade relatively rapidly within the body of a mammal, thus detracting from their usefulness in certain procedures where long-term presence of intact particles. On the other hand, degradation of prior art materials can release toxic or irritating compounds that cause adverse reactions in patients.

It is also a problem in the art for certain types of prior art particles that it is difficult to achieve desirable suspension properties when the particles are incorporated into a delivery suspension for injection at a site in the body to be treated. Many times, the particles settle or tend to float in the solution in such a way that they are not uniformly suspended for regular delivery. Additionally, the particles may tend to aggregate in the delivery solution and / or adhere to some part of the delivery device, making it necessary to compensate for these attractive / adhesive forces.

In order to achieve a stable dispersion, it is known to add suitable dispersing agents which may include surfactant agents aimed at breaking the attractive interaction of particles. Depending on the nature of the particle interaction, the following materials can be used: cationic, anionic or non-ionic surfactants such as Tween ™ 20, Tween ™ 40, Tween ™ 80, polyethylene glycols, sodium dodecyl sulfate, various source proteins natural such as serum albumin, or any other macromolecular surfactants in the delivery formulation. Additionally, thickening agents can be used to help prevent the particles from settling and to increase the viscosity of the solution, for example, polyvinyl alcohols, polyvinyl pyrrolidones, sugars or dextrins. Density additives can also be used to achieve buoyancy.

It can also be difficult to visualize microparticles in solution to determine their degree of suspension when using clear, transparent polymeric acrylate hydrogel beads in aqueous suspension. Attempts to use the inert precipitate, barium sulfate, in particulate form is known as an additive for bone cement, for silicones for rendering the elements visible during X-ray examination, and for providing radiopacity to polymeric acrylate particles. . See Jayakrishnan et al., Bull. Mat. Sci., Vol. 12, No. 1, pp. 17-25 (1989). Barium sulfate is also known to improve fluidization, and is frequently used as an inorganic filler to impart non-stick behavior to wet, aggregated particles. Other prior art attempts to increase the visualization of microparticles include the use of gold, for example Embosphere Gold ™ provides a magenta color to acrylate microparticles using small amounts of gold.

In certain medical applications, it may also be of value to provide microparticles such as microspheres in one or more sizes. Additionally, it may also be of value for a user to provide each such incorporated microsphere size with colorants associated with color coding to indicate the size of the microspheres to the user. In still other applications of use, it may be of further value to provide sized and color-coded microspheres to a user in similarly color-coded syringes or other containers for transport and supply to further assist a user in identification. the size of the microspheres used.

There is thus a need in the art for small particles that can be formed to have a generally spherical preferential configuration for certain applications such as various therapeutic and diagnostic procedures that are not degraded by the natural systems of the mammalian system, are biocompatible, are easy to display in suspension while in use and / or demonstrate acceptable suspension and physical properties.

ES 2 564 151 T3

US2006 / 0088476 describes particles for use in therapeutic and / or diagnostic procedures. The particles include poly [bis (trifluoroethoxy) phosphazene] and / or a derivative thereof that may be present throughout the particles or within an outer coating of the particles. The particles can also include a core having a hydrogel formed from an acrylic-based polymer. The barium sulfate can also be provided to the core of the particles as a coating or absorbed within the core of the particles. The particles can be used to minimize blood flow to mammalian tissues by occluding at least a portion of a mammalian blood vessel, or to deliver an active agent to a localized area within a mammalian body by contact. of a localized area with at least one of the particles. Furthermore, the particles are useful in sustained release formulations that include active agents for oral administration, in the form of tracer particles for injection into the bloodstream of a mammal or for use in enhanced ultrasound imaging. The particles may include density increasing agents to achieve useful levels of buoyancy in suspension.

Brief summary of the invention

According to a first aspect of the present invention, color-coded polymeric particles are provided according to claims 1 to 9. According to a second aspect of the invention, a method is provided for making color-coded polymeric particles of According to claims 10 to 14. Also described herein is a particle for use in a therapeutic and / or diagnostic procedure. The particle comprises poly [bis (trifluoroethoxy) phosphazene] and / or a derivative thereof.

Also described herein are particles comprising poly [bis (trifluoroethoxy) phosphazene and / or a derivative thereof provided as microspheres provided in one or more specified sizes.

Also described herein are particles comprising poly [bis (trifluoroethoxy) phosphazene and / or a derivative thereof provided as sized microspheres and further comprising a color-coded dye incorporated into or attached to the exterior of the microspheres to visually aid a user. in identifying the size of microspheres in use.

The microspheres described herein may further be provided as sized microspheres further comprising a color-coded dye incorporated into or attached to the exterior of the microspheres and contained or supplied in a similarly color-coded syringe or other shipping or delivery container. to further visually assist a user in providing visual confirmation of the specific size of microspheres in use.

Also described herein is a method of minimizing blood flow to a tissue in a mammal which comprises occluding at least a portion of a blood vessel of the mammal with at least one particle, wherein the particle comprises a poly [bis (trifluoroethoxy ) phosphazene] and / or a derivative thereof.

Further described herein is a method of delivering an active agent to a localized area within a mammalian body, comprising contacting the localized area with at least one of a particle comprising poly [bis (trifluoroethoxy) phosphazene] and / or or a derivative thereof and an active agent, such that an effective amount of the active agent is exposed to the localized area.

Also described herein is a sustained release formulation of an active agent for oral administration, the formulation comprising a polymer capsule and an active agent, wherein the polymeric capsule comprises poly [bis (trifluoroethoxy) phosphazene] and / or a derivative of same.

Also described herein is a method for tracking the passage of a particle through a blood vessel in a mammal, the method comprising injection into the bloodstream of a mammal of at least one tracer particle, the tracer particle comprising poly [bis ( trifluoroethoxy) phosphazene] and / or a derivative thereof and a contrast agent, and particle pathway imaging.

Additionally, an enhanced method of ultrasound imaging is described herein. The method comprises administering to an ultrasound subject at least one hollow microcapsule comprising poly [bis (trifluoroethoxy) phosphazene] and / or a derivative thereof to an area of ​​the ultrasound subject, and imaging the subject area using ultrasound.

Also described herein is a method of delivering an active agent to a localized area in the body of a mammal which comprises contacting the localized area with at least one of a particle comprising poly [bis (trifluoroethoxy) phosphazene] and / or a derivative thereof and an active agent, such that an effective amount of the active agent is exposed to the localized area, wherein the particle comprises a density increasing agent.

Furthermore, a method for minimizing agglomeration of the particles formed from acrylic-based polymers is described in which the method comprises providing barium sulfate to the core and / or the surface of the particles.

ES 2 564 151 T3

Brief description of the various views of the drawings

The above summary, as well as the following detailed description of the invention, will be better understood when read in conjunction with the accompanying drawings. For the purposes of illustrating the invention, embodiments that are presently preferred are shown in the drawings. It should be understood, however, that the invention is not limited to the precise arrangements and instrumentalities shown.

In the drawings:

Figure 1 shows a schematic representation of a general cryoextraction scheme used to prepare the particles according to one embodiment of the invention;

Figure 2 shows the manual dripping technique by which the polymer solution was supplied to liquid nitrogen in the preparation of the microspheres of Example 1, here;

Figure 3A shows representative color-coded, different-sized A, B, and C microspheres of the present invention.

Figure 3B shows a cross-sectional drawing of a conceptual blood vessel with arrows indicating the direction of blood flow, where the blood vessel narrows from a larger proximal diameter to a smaller distal diameter, and where microspheres of size different and color-coded components of the present invention have been injected sequentially in ascending order of size to occlude the vessel.

Figure 3C shows a syringe containing microspheres of the present invention where the microspheres are sized and color-coded to indicate their size, and where the syringe is also color-coded similar to facilitate user identification and verification of microspheres sized in use.

Detailed description of the invention

Described herein are particles that can be manufactured using poly [bis (trifluoroethoxy) phosphazene] and / or derivatives thereof, as well as methods of preparing such particles. Additionally, therapeutic and / or diagnostic methods and procedures using the particles are described herein as described herein, including methods of embolization using the particles, methods of delivering an active agent using the particle (either orally or locally), the methods of tracking or visualizing blood or other biological fluids through the body using the particles, and enhanced ultrasound (sonography) methods using the particles.

Also included are sustained release drug delivery formulations for oral administration, including particles for localized delivery of an active agent to the gastrointestinal system and / or systemic delivery of an active agent, as well as a drug delivery formulation of sustained release that can be injected subcutaneously or intravenously for localized delivery of an active agent.

All methods, compositions and formulations of the invention use at least one particle as described herein. Particle and particle as used herein mean a substantially spherical or ellipsoid, hollow or solid article (s), which may have any suitable diameter for use in the specific methods and applications described below, including a microsphere (s) ( s) and a nanosphere (s), beads and other bodies of a similar nature known in the art.

That of the preferred particles of the invention according to an embodiment described herein is composed, in whole or in part, of the specific polyphosphazene polymer known as poly [bis (trifluoroethoxy) phosphazene] or a derivative of poly [bis (trifluoroethoxy) phosphazene]. The use of this specific polymer provides particles that are at least in part inorganic in that they include an inorganic polymer backbone and that are also biocompatible in that when introduced into a mammal (including humans and animals), they do not significantly induce a response from the specific or non-specific immune systems. The particles can be used as controlled drug delivery vehicles or tracer particles for visualization of blood vessels and other organs.

The particles are useful in a variety of therapeutic and / or diagnostic procedures in part because they can be prepared in sizes large enough to occlude a blood vessel as well as small enough to easily pass through smaller vessels. , for example, display or drug delivery purposes. Additionally, due to the biocompatible nature of the polymer, the particles facilitate the avoidance or elimination of immunogenic reactions generally encountered when foreign bodies are introduced into a mammalian body, such as implant rejection or allergic shock ”, and other adverse reactions of the immune system. On the other hand, it has been found that the particles of the invention exhibit reduced biodegradation in vivo, which increases the long-term stability of the particle in the biological environment. Furthermore, in those situations where some degradation is suffered by the polymer in the particle, the products released from the degradation include only non-toxic concentrations of phosphorus, ammonia and

ES 2 564 151 T3 trifluoroethanol, which, advantageously, is known to promote anti-inflammatory responses when in contact with mammalian tissue.

The layer of each particle in the invention is formed at least in part from the polymer, poly [bis {2,2,2-trifluoroethoxy) phosphazene] or a derivative thereof (further referred to herein as poly [bis (trifluoroethoxy) phosphazene]. As described herein, the poly [bis (2,2,2-trifluoroethoxy) phosphazene] polymer or derivatives thereof have chemical and biological qualities that distinguish this polymer from other known polymers in general, and from other known polyphosphazenes in particular. In one aspect of this invention, the polyphosphazene is poly [bis (2,2,2-trifluoroethoxy) phosphazene] or derivatives thereof, such as other alkoxide, halogenated alkoxide, or substituted fluorinated alkoxide analogues thereof. Poly [bis (trifluoroethoxy) phosphazene] polymer is composed of repeating monomers represented by formula (I) shown below:

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(I) where R¹ to R⁶ they are all trifluoroethoxy groups (OCH2CF3), and where n can range from at least about 40 to about 100,000, as disclosed herein. Alternatively, derivatives of this polymer can be used in the present invention. The term derivative or derivatives is understood to refer to polymers formed from monomers having the structure of formula I but where one or more of the functional groups R¹ to R⁶ is replaced by a different functional group (s), such as an unsubstituted alkoxide, a halogenated alkoxide, a fluorinated alkoxide, or any combination thereof, or where one or more of the R¹ to R⁶ it is replaced by any of the other functional groups disclosed herein, but where the biological inertness of the polymer is not substantially altered.

In one aspect of the polyphosphazene of formula (I) illustrated above, for example, at least one of the substituents R¹to R⁶ it can be an unsubstituted alkoxy substituent, such as methoxy (OCH3), ethoxy (OCH2CH3), or n-propoxy (OCH2CH2CH3). In another aspect, for example, at least one of the R substituents¹ to R⁶ it is an alkoxy group substituted with at least one fluorine atom. Examples of Useful Fluorine Substituted Alkoxy Groups R¹ to R⁶ include, but are not limited to, OCF₃, OCH2CF3, OCH₂CH₂CF₃, OCH₂CF₂CF₃, OCH (CF₃)₂, OCCH₃(CF₃)₂, OCH2CF2CF2CF3, OCH₂(CF₂) 3CF₃, OCH₂(CF₂) 4CF₃, OCH₂(CF₂) sCF₃, OCH2 (CF₂)₆CF3, OCH₂(CF₂) 7CF₃, OCH2CF2CHF2, OCH2CF2CF2CHF2, OCH₂(CF₂) 3CHF₂, OCH₂(CF₂) 4CHF₂, OCH2 (CF₂)₅CHF2, OCH2 (CF₂)₆CHF2, OCH₂(CF₂) 7CHF₂, and the like. Thus, while trifluoroethoxy groups (OCH2CF3) are preferred, these additional exemplary functional groups can also be used alone, in combination with trifluoroethoxy, or in combination with one another. In one aspect, examples of especially useful fluorinated alkoxy functional groups that may be used include, but are not limited to, 2,2,3,3,3-pentafluoropropyloxy (OCH2CF2CF3), 2,2,2,2'2'2 ' -hexafluoroisopropyloxy (OCH (CF3) 2),

2,2,3,3,4,4,4-heptafluorobutyloxy (OCH2CF2CF2CF3), 3,3,4,4,5,5,6,6,7,7,8,8,8-tridecafluorooctyloxy (OCH₂(CF₂) 7CF₃), 2,2,3,3, -tetrafluoropropyloxy (OCH₂CF₂CHF₂), 2,2,3,3,4,4-hexafluorobutyloxy (OCH2CF2CF2CHF2),

3,3,4,4,5,5,6,6,7,7,8,8-dodecafluorooctyloxy (OCH2 (CF2) 7CHF2), and the like, including combinations thereof.

Furthermore, in some embodiments, 1% or less of the R groups¹ to R⁶ they can be alkenoxy groups, a feature that can aid in crosslinking to provide a more elastomeric phosphazene polymer. In this aspect, alkenoxy groups include OCH2CH = CH2, OCH2CH2CH = CH2, allylphenoxy groups, and the like, including combinations thereof. Also in formula (I) illustrated here, the residues R¹ to R⁶ they are each independently variable and therefore can be the same or different.

So it indicates that n can be large as ⁰⁰ In Formula I, it is intended to specify the values ​​of n encompassing polyphosphazene polymers that can have an average molecular weight of up to about 75 million Daltons. For example, in one aspect, n can range from at least about 40 to about 100,000. In another aspect, by indicating that n can be as large as⁰⁰ In Formula I, it is intended to specify values ​​of n from about 4,000 to about 50,000, more preferably, n is from about 7,000 to about 40,000, and most preferably n is about 13,000 to about 30,000.

In another aspect of this invention, the polymer used to prepare the polymers disclosed herein have a molecular weight based on the above formula, which may be a molecular weight of at least about 70,000 g / mole, more preferably at least about 1,000,000 g / mole. mol, and still more preferably a molecular weight of at least about 3x10® g / mol to about 20x10® gmol. Most preferred are polymers having molecular weights of at least about 10,000,000 g / mol.

ES 2 564 151 T3

In a further aspect of the polyphosphazene formula (1) illustrated here, n is 2 a ~, and R¹ to R⁶ are groups that are each independently selected from alkyl, aminoalkyl, haloalkyl, thioalkyl, thioaryl, alkoxy, haloalkoxy, aryloxy, haloaryloxy, alkylthiolate, arylthiolate. alkylsulfonyl, alkylamino, dialkylamino, heterocycloalkyl comprising one or more heteroatoms selected from nitrogen, oxygen, sulfur, phosphorus, or a combination thereof, or heteroaryl comprising one or more heteroatoms selected from nitrogen, oxygen, sulfur, phosphorus, or a combination thereof. In this aspect of formula (I), the pendant side groups or structural units (also called residues) R¹ to R⁶ they are each independently variable and therefore can be the same or different. Furthermore, R¹ to R⁶ it can be substituted or unsubstituted. Alkyl groups or structural units within alkoxy, alkylsulfonyl, dialkylamino, and other alkyl-containing groups can be, for example, straight or branched chain alkyl groups having 1 to 20 carbon atoms, typically 1 to 12 carbon atoms. carbon, it being possible for alkyl groups to be further substituted, for example, by at least one halogen atom, such as a fluorine atom or other functional group, such as those indicated for R groups¹ to R⁶previous. By specifying alkyl groups such as propyl or butyl, they are intended to encompass any particular alkyl group isomer.

In one aspect, examples of alkoxy groups include methoxy, ethoxy, propoxy, and butoxy, and the like, which can also be further substituted. For example, the alkoxy group can be substituted by at least one fluorine atom, with 2,2,2-trifluoroethoxy constituting a useful alkoxy group. In another aspect, one or more of the alkoxy groups contain at least one fluorine atom. Furthermore, the alkoxy group can contain at least two fluorine atoms or the alkoxy group can contain three fluorine atoms. For example, the polyphosphazene that is combined with the silicone can be poly [bis (2,2,2-trifluoroethoxy) phosphazene]. The alkoxy groups of the polymer can also be combinations of the aforementioned embodiments wherein one or more fluorine atoms are present in the polyphosphazene in combination with other groups or atoms.

Examples of alkylsulfonyl substituents include methylsulfonyl, ethylsulfonyl, propylsulfonyl, and butylsulfonyl groups. Examples of dialkylamino substituents include, but are not limited to, dimethyl-, diethyl-, dipropyl, and dibutylamino groups. Again, by specifying alkyl groups such as propyl or butyl, it is intended to encompass any isomer of the particular alkyl group, exemplary aryloxy groups include, for example, compounds having one or more aromatic ring systems having at least one atom. oxygen, non-oxygen atom, and / or rings having alkoxy substituents, it being possible for the aryl group to be substituted for example by at least one alkyl or alkoxy substituent defined above. Examples of aryloxy groups include phenoxy and naphthyl groups, and derivatives thereof including, for example, substituted phenoxy and naphthyl groups.

The heterocycloalkyl group can be, for example, a ring system containing from 3 to 10 atoms, at least one ring atom which is a nitrogen, oxygen, sulfur, phosphorus atom, or any combination of these heteroatoms. The heterocycloalkyl group can be substituted, for example, by at least one alkyl or alkoxy substituent as defined above. Examples of heterocycloalkyl groups include piperidinyl, piperazinyl, pyrrolidinyl, and morpholinyl groups, and substituted analogs thereof.

The heteroaryl group can be, for example, a compound having one or more aromatic ring systems, at least one ring atom which is a nitrogen, an oxygen, a sulfur, a phosphor, or any combination of these heteroatoms. The heteroaryl group can be substituted for example by at least one alkyl or alkoxy substituent defined above. Examples of heteroaryl groups include imidazolyl, thiophene, furan, oxazolyl, pyrrolyl, pyridinyl, pyridine, isoquinoline, and quinolinyl groups, and derivatives thereof, such as substituted groups.

The diameter of a particle formed in accordance with the invention will vary depending on the end application in which the particle is used. The diameter of such particles is preferably from about 1 to about 5,000 µm, with a diameter of from about 1 to about 1,000 µm being most preferred. Other preferred sizes include diameters from about 200 to about 500 µm, about 1 to about 200 µm, and greater than about 500 µm. In particle methods where more than one particle is preferred, it is not necessary that all particles be of the same diameter or shape. In one aspect, the polymeric particles are substantially uniform in size, which means that the size of the particles can be determined by the process by which they are prepared and isolated, and are characterized by a narrow size distribution. By substantially uniform in size, it is generally intended to reflect that the particle size in accordance with the design specification may vary from less than or equal to about ± 5%, less than or equal to about ± 10%, less than or equal to approximately ± 15%, less than or equal to approximately ± 20%, less than or equal to approximately ± 25%, less than or equal to approximately ± 30%, or less than or equal to approximately ± 35% of design specification . In one aspect, for example, particle size distributions described herein may be narrower as the design specification of the particle to be manufactured becomes larger. For example, particles between about 700 pm and about 1,000 pm can vary from less than or equal to only about ± 3 to 5% from the design specification, while particles between about 40 pm and about 100 pm they may vary less than or equal to approximately ± 20-25% from the design specification.

ES 2 564 151 T3

The particles may also include other compounds that function to enhance, alter or otherwise modify the behavior of the polymer or particle, either during its preparation or in its therapeutic and / or diagnostic use. For example, active agents such as peptides, proteins, hormones, carbohydrates, polysaccharides, nucleic acids, lipids, vitamins, steroids, and organic or inorganic drugs can be incorporated into the particle. Excipients such as dextran, other sugars, polyethylene glycol, glucose, and various salts, including, for example, chitosan glutamate, can be included in the particle.

Additionally, if desired, polymers other than poly [bis (trifluoroethoxy) phosphazene] and / or its derivative can be included with the particle. Examples of polymers can include poly (lactic acid), poly (lactic-co-glycolic acid), poly (caprolactone), polycarbonates, polyamides, polyanhydrides, polyamino acids, polyorthoesters, polyacetals, polycyanoacrylate, and polyurethanes. Other copolymers include polyacrylates, ethylene vinyl acetate copolymers, acyl substituted cellulose acetates and derivatives thereof, degradable or non-degradable polyurethanes, polystyrenes, polyvinyl chloride, polyvinyl fluoride, poly (vinyl imidazole), chlorosulfonated polyolefins. , and polyethylene oxide. Examples of polyacrylates include acrylic acid, butyl acrylate, ethylhexyl acrylate, methyl acrylate, ethyl acrylate, acrylonitrile, methyl methacrylate, TMPTA (trimethylolpropane triacrylate), and the like. Selected compounds can be incorporated by any means known in the art, including diffusing, inserting, or entrapment of the additional compounds in the matrix of an already formed particle or by adding the additional compound to the polymer melt or to a polymer solvent in the preparation of the particle as described here.

The charged or discharged particle may be coated with an additional polymer layer or layers, including polymers such as those mentioned above. PTFEP or its derivatives are used to form a coating on a particle formed from polymers or copolymers that are used to form particles as described herein. PTFEP is applied as a coating on a microparticle (s) formed of an animic acrylic based polymer as discussed in more detail below.

Coatings are beneficial, for example, if the particles are to be used in a sustained release, orally administered, drug delivery formulation (enteric coating) or if the particles are to be loaded with a potentially toxic contrast agent ( non-biodegradable coating).

Microspheres can be prepared by any means known in the art that is suitable for the preparation of particles containing poly [bis (trifluoroethoxy) phosphazene]. In a process according to an embodiment herein a polymer solution is prepared by mixing one or more polymer solvents and the PTFE and / or a derivative thereof until the polymer dissolves,

Suitable solvents for use in preparing the polymer solution include any in which the PTFEP polymer and / or its derivatives are soluble. Examples of solvents include, without limitation, ethyl-, propyl-, butyl-, pentyl, octylacetate, acetone, methyl ethyl ketone, methylpropyl ketone, methyl isobutyl ketone, tetrahydrofuran, cyclohexanone, dimethylacetamide, acetonitrile, dimethyl ether, hexafluorobenzene, or combinations thereof.

The polymer solution contains the PTFEP and / or its derived polymer in a concentration of from about 1% by weight of polymer to 20% by weight of polymer, preferably from about 5% to 10% by weight of polymer. Other polymers, as discussed above, can be present in the solution, or can be added to the container in the form of a second solution powder or other form, if it is desired to include such polymers in the final particle.

In performing the process, the polymer solution is then dispensed, preferably in the form of drops or an aerosol, into a container containing a non-solvent. By non-solvent is meant any of the organic or inorganic solvents that do not substantially dissolve the PTFEP polymer and that have a melting point that is lower relative to the melting point of the solvent in which the polymer is dissolved (solvent of polymer), such that the non-solvent thaws before the solvent thaws in the course of the incubation step. Preferably, this difference between the melting point of the non-solvent and the polymer solvent is about 10 ° C, more preferably about 15 ° C, and most preferably, greater than about 20 ° C. Under certain conditions, it has been found that the structural integrity of the resulting particle can be enhanced if the difference of the melting points of the polymer solvent and the non-solvent is greater than 15 ° C. However, it is sufficient that the point of the non-solvent is more than slightly lower than that of the polymer solvent.

The non-solvent / polymer solvent combination is incubated for approximately 1 to 5 days or until the polymer solvent has been completely removed from the particles. Although not wishing to be bound by theory, it was hypothesized that, during incubation, the non-solvent functions to extract the polymer solvent from the microscopic droplets of polymer solution of the particles such that the polymer is at less gelled. As the incubation period passes, the droplets will shrink and the solvent is further removed, leading to a hardened outer polymeric layer containing a gelled polymer core and finally, after completion of the incubation, a complete removal of residual solvent. . To ensure that the polymeric droplets retain a substantially spherical shape during the incubation period, they are kept in a frozen or substantially gelled state for most if not the entire period of

ES 2 564 151 T3 incubation. Therefore, the temperature of the non-solvent can remain below the melting point of the solvent during the cryoextraction process.

As shown in Figure 1, in the container marked (a), the droplets of polymer solution are shown being dispensed with either a syringe or other device to a controlled rat over a top layer of liquid nitrogen. The nitrogen layer is located over a lower layer consisting of the selected non-solvent, which will eventually serve to extract the solvent from the frozen polymer solution droplets. The non-solvent layer has been pre-frozen with liquid nitrogen prior to dispensing the polymer solution. The container marked (b) shows the onset of condensation of the frozen non-solvent, into which the frozen polymeric droplets will sink. The container marked (c) shows the cryoextraction procedure after approximately three days of incubation where the droplets of polymer solution, incubated in the non-solvent, have been depleted of a substantial amount of solvent. The result is a gelled polymeric particle in the form of a bead that has a hardened outer layer. As can be seen from the representation, the height of the non-solvent within the container is slightly reduced due to some evaporation of the non-solvent. The size of the beads will shrink substantially during this process depending on the initial concentration of the polymer in the polymer solution.

In one embodiment of a method of preparing a PTFEP-containing particle according to the invention, such particles can be formed using any way known or developed in the art. Two example preferred methods that accomplish this include where (i) the non-solvent resident in the container in performing the method described above is cooled to near its freezing point or to its freezing point prior to the addition of the solution of polymer such that the polymer droplets freeze upon contact with the precooled non-solvent; or (ii) the polymer droplets are frozen by contacting them with a liquefied gas, such as nitrogen, which is placed on a bed of pre-frozen non-solvent (see, Figure 2). In method (ii), after the nitrogen evaporates, the non-solvent is slowly thawed and the microspheres in their frozen state will sink into the liquid, cold non-solvent where the extraction process will take place (removal of the solvent polymer).

By modifying this general process, particles that are hollow or substantially hollow or porous can be prepared. For example, if removal of the solvent from the beads is carried out quickly, for example by applying a vacuum during the final stage of the incubation, it will result in porous beads.

The particles of the invention can be prepared in any desired size. Microspheres can be obtained by nebulizing the polymer solution into a polymer aerosol using either pneumatic or ultrasonic nozzles, such as, for example, a Sonotek 8700-60ms or a Lechler US50 ultrasonic nozzle, each available from Sono [.tek] Corporation, Milton, New York, USA and Lechler GmbH, Metzingen, Germany. Larger particles can be obtained by dispensing the droplets into the non-solvent solution using a syringe or other dropping device. Furthermore, as will be known to a person skilled in the art, the size of the particle can also be altered or modified by an increase or decrease in the initial concentration of the polymer in the polymer solution, such as a higher concentration will lead to a increase in the diameter of the sphere.

The particles disclosed herein include a standard and / or preferred core based on an acrylic polymer or copolymer with a PTFEP layer. Such particles can provide a preferred spherical shape and improved specific gravity for use in an embolization contrast media suspension. The acrylic polymer-based polymers with the PTFEP layer described herein provide substantially spherical shape, mechanical flexibility and compressibility, improved gravity specific properties. Core polymers can be formed using any acceptable technique known in the art, such as that described in B. Thanoo et al., Preparation of Hydrogel Beads from Crosslinked Poly (Methyl Methacrylate) Microspheres by Alkaline Hydrolysis, J. Appl. P. Sci., Vol. 38, 11531161 (1990). Such acrylic-based polymers are preferably formed by the polymerization of unhydrolyzed precursors, including methyl acrylate (MA), methyl methacrylate (MMA), ethyl methacrylate (EMA), hexamethyl (HMMA), or hydroxyethyl methacrylate (HEMA), and derivatives, variants or copolymers of such acrylic acid derivatives. The most preferred is MMA. The polymer is present in the core in a hydrated or partially hydrated form (hydrogel). Such polymers are preferably crosslinked in order to provide suitable hydrogel structure and properties, such as enhanced non-biodegradability, and to help retain the mechanical stability of the polymer structure by resisting dissolution by water.

Preferably, the core prepolymers are formed by dispersion polymerization which can be of the suspension or emulsion polymerization type. The emulsion polymerization results in substantially spherical core particles from about 10 nm to about 10 microns. Suspension polymerization will result in similar but larger particles of about 50 to about 1200 microns.

Suspension polymerization can be initiated with a thermal initiator, which can be solubilized in the aqueous phase or, more preferably, in the monomer phase. Suitable initiators for use in the monomer phase composition include benzoyl peroxide, lauroyl peroxide, or other similar peroxide-based initiators.

ES 2 564 151 T3 known or to be developed in the art, the most preferred initiator being lauroyl peroxide. The initiator is preferably present in an amount of from about 0.1 to about 5 percent by weight based on the weight of the monomer, more preferably from about 0.3 to about 1 percent by weight based on the weight of the monomer. As noted above, a crosslinking comonomer is preferred for use in forming the hydrated polymer. Crosslinking comonomers suitable for use with the acrylic-based principle monomer used in the preparation of a polymerized particle core, include various glycol-based materials such as ethylene glycol dimethacrylate (EGDMA), diethylene glycol dimethacrylate (DEGDMA), or the most preferably, triethylene glycol dimethacrylate (TEGDMA). A chain transfer agent can also be provided, if desired. Any suitable MA polymerization chain transfer agent can be used. In the preferred embodiment herein, dodecyl mercaptan can be used as a chain transfer agent in acceptable amounts for the particular polymerization reaction.

The composition of the aqueous phase preferably includes a surfactant / dispersant, as well as a complexing agent, and an optional regulator is necessary. Surfactants / dispersants must be compatible with the monomers used herein, including Cyanamer® 370M, polyacrylic acid, and partially hydrolyzed polyvinyl alcohol surfactants, such as 4/88, 26/88, 40/88. A dispersant must be present in an amount of about 0.1 to about 5 weight percent based on the amount of water in the dispersion, more preferably about 0.2 to about 1 cient by weight based on the amount of water in dispersion. An optional buffer solution can be used, if necessary, to maintain proper pH. A preferred buffer solution includes sodium phosphates (Na2HPÜ4 / NaH2PO4). A suitable complexing agent is ethylenediaminetetraacetic acid (EDTA), which can be added to the aqueous phase in a concentration of from about 10 to about 40 ppm EDTA, and more preferably about 20 to about 30 ppm. It is preferred that in the aqueous phase composition, the ratio of monomer to water is from about 1: 4 to about 1: 6.

Polymerization should take place at about ambient conditions, preferably greater than 60 ° C to about 80 ° C with a gel time of about one to two hours. Agitation at 100 to 500 rpm rats is preferred for core particle formation, with fewer rates applying to the larger core particles and higher rates applying to the smaller core particles.

Once PMMA core particles are formed, such as microparticles, they are preferably subjected to hydrolysis conditions typical of those in the art, including the use of about 1-10 molar excess of potassium hydroxide per mole of PMMA. Such potassium hydroxide is provided in a hydroxide concentration of about 1-15% potassium hydroxide in ethylene glycol. The solution is then preferably heated to temperatures of about 150-185 ° C for several hours. Alternatively, to minimize reagent amounts and cost, it is preferred that lower amounts of potassium hydroxide are used which are less than about 5 molar excess of potassium hydroxide per mole of PMMA, more preferably about 3 molar excess. or less. For such hydrolytic reactions, a concentration of about 10-15% potassium hydroxide in ethylene glycol is also preferably used, and more preferably about 14% to about 15%. It will be understood by one skilled in the art that heating conditions at higher temperatures can be used to decrease overall reaction times. Reaction times can vary depending on the total diameter of the resulting core particles. For example, the following conditions are capable of providing particles that have approximately 35% compressibility and the desired stability: for diameters of approximately 200-300 pm, the solution should be heated for approximately 7.5 to approximately 8.5 hours, for diameters of approximately 300- 355 pm, approximately 9.5 to approximately 10.5 hours; for diameters of about 355-400 pm, about 11.5 to about 12.5 hours; and for about 400 to 455 pm, about 13.5 to about 14.5 hours, etc. The particle size of the core can be adjusted using variations in the polymerization process, for example, by varying the stirring speed and the ratio of the monomer to the aqueous phase. Also, smaller sizes can be achieved by increasing the surfactant / dispersant ratio.

After hydrolysis, the core particles are separated from the reaction mixture and their pH can be adjusted to any range, as appropriate for additional processing steps or intended uses. The pH of the particle core can be adjusted from about 1.0 to about 9.4, preferably about 7.4 if intended for physiological application. Since the size, swelling ratio and elasticity of the hydrogel core material are dependent on pH value, lower pH values ​​can be used to have beneficial effects during drying to avoid particle agglomeration and / or damage. structural. The core particles are preferably screened in different size fractions according to the intended use. Drying of the particles preferably occurs using any standard drying process, including the use of an oven at a temperature of about 40 ° -80 ° C for several hours to about a day.

To provide desired surface properties to the hydrophilic hydrogel core particles, in order to provide adhesion for the reception of a PTFEP coating, the surface of the hydrogel may be treated with any suitable ionic or non-ionic surfactant, such as tetraalkylammonium salts, polyalcohols and materials

ES 2 564 151 T3 similar. A more permanent change in adhesion properties occurs by rendering the surface of the hydrophobic core particles by reacting their polymethacrylic acid groups with a suitable reagent. Suitable reagents include, but are not limited to, hydrophobic alcohols, amides, and carboxylic acid derivatives, more preferably they include halogenated alcohols such as trifluoroethanol. Such surface treatment also prevents delamination of the core coating once the coating is applied. Preferred surface treatments may include, without limitation, an initial treatment with thionyl chloride followed by reaction with trifluoroethanol. Alternatively, the surface can be treated by suspending the core particles in a mixture of sulfuric acid and a hydrophobic alcohol, such as trifluoroethanol. Such treatments are preferred if the core particles are to be coated as long as they minimize any delamination of a coating.

Alternatively, and more preferably, the PMA core particles can be coated with a surface layer of and / or infused with barium sulfate. Barium sulfate is radiopaque and aids in the visualization of finished particles when in use. It also provides enhanced fluidization properties to the particles such that it reduces agglomeration especially during drying and allows fluid bed coating of the PMA core particles with an outer PTFEP coating, thereby providing improved adhesion between a PTFEP outer core and polymeric acrylate core particles. By allowing fluidization even when the core particles are swollen, barium sulfate also improves overall coating and adhesion properties. By allowing coating of the core particles, even in a swollen state with PTFEP, barium sulfate also reduces the potential tendency of the PTFEP layer to crack or break, compared to coating the core particles in a dry and subsequently exposing the particles to a suspension in which the core particles swell and exert force on the PTFEP layer. A barium sulfate coating on the core particles is preferably applied by adhering the barium sulfate in the form of an opaque coating to the hydrogel surface of the PMA beads. Barium sulfate can also help reduce electrostatic effects that limit particle size. By allowing additional moisture absorption, barium sulfate tends to counteract electrostatic effects.

Barium sulfate crystals that adhere only poorly to PMA core particles can be covalently crosslinked or chemically grafted to the surface of the core particle by spray coating a sufficient amount of an aminosilane adhesion promoter. on the PMA core particle. This will help to effectively reduce the barium sulfate particulate matter in solution after hydration of the core particles. Exemplary core particles include 3-aminopropyltrimethoxysilane and similar silane-based adhesion promoters.

To improve visualization of the microparticles made as noted herein, the absorption of a water soluble organic dye into the hydrogel core particles is included. Exemplary dyes are preferably those dyes approved by the FDA for human use and which are known or developed for safe, non-toxic use in the body and which are capable of providing acceptable contrast. Organic colorants can include colorants such as D&amp;C Violet No. 2 and others preferably approved for medical device uses, such as for contact lenses and absorbable sutures. While barium sulfate operates as a finely dispersed pigment and inorganic filler that makes the particles visible by light diffraction due to the small crystal size, the colorants when impregnated into the particles absorb the complementary part of the spectrum. visible color.

The microparticles made according to the above process to form a core hydrogel polymer are then coated with PTFEP and / or its derivatives. Any suitable coating process can be used, including solvent fluidized bed and / or spray techniques. However, preferred results can be achieved using fluidized bed techniques in which the core particles pass through an air stream and are coated through spray while rotating within the air stream. The PTFE or derivative polymer is provided in dilute solution for spraying to prevent clogging of the nozzle.

Examples of solvents for use in such solutions include ethyl acetate, acetone, hexafluorobenzene, methyl ethyl ketone, and the like solvents and mixtures and combinations thereof, most preferred being ethyl acetate alone or in combination with isoamyl acetate. Typical preferred concentrations include about 0.01 to about 0.3 weight percent PTFEP or its derivative in solution, more preferably about 0.02 to 0.2 weight percent PTFEP, and most preferably about 0.075 to about 0.2 weight percent. It should be understood based on this disclosure that the type of hydrogel core can be varied as can the technique for coating a core particle, however a core that is useful in treatment techniques and applications is preferred. described herein is formed and subsequently coated with PTFEP and / or its derivatives as described herein.

As previously discussed, the particles can be used in various medical and therapeutic applications, such as embolization, drug delivery, (ultrasound) imaging, and as tracer particles. For example, in one embodiment, the invention includes particles for use in a method of minimizing blood flow to a specific tissue in a mammal. This process, commonly known as embolization, includes the occlusion or obstruction of at least part of a vessel, or the entire vessel, with one or more of the

ES 2 564 151 T3 particles of the invention. Such a procedure is particularly useful in the treatment of diseases and pathologies that involve undesirable vascularized tissues, for example, tumor tissue or disorders that involve the uncontrolled proliferation of certain cells, such as endometriosis. In such procedures, the particles are prepared according to the procedures described above, and can be inserted into the blood vessel by any known invasive or non-invasive medical practice or to be developed in the art, such as through a catheter, a syringe. , or a surgical incision. Embolization can be carried out in such a way that only a part of the blood vessel is occluded, or the entire vessel can be occluded. In the method, if desired, particles that have been loaded with an active agent such as a cytostatic agent, an anti-inflammatory agent, an antimitogenic or cell proliferation active agent, a hormone, or any other desirable active agent, such as described here. The embolization particles according to the present invention are capable of demonstrating improved optical visibility, additional radiopacity, and an optimal specific density of approximately 1.17 g / cm³. The embolization particles in this invention can be used with different dyes as markers as indicated above for particle sizes, embedded pharmaceuticals for localized drug delivery, and controlled drug elution characteristics.

For use in embolization therapy, particle density is preferably taken into consideration to ensure beneficial particle delivery properties. Possible clogging of a catheter-based delivery system can occur if a non-matching delivery medium in density is used. In addition, it is desirable to include a certain minimal amount of contrast agent in the delivery medium to achieve sufficient levels of fluoroscopic contrast during surgery. Currently, the density of polymethacrylate hydrogel is between 1.05 g / cm³ and 1.10 g / cm³ depending on the equilibrium water content. The most common non-ionic iodinated contrast agent media with 300 mg iodine per ml have densities of 1.32-1.34 g / cm³. As used herein, buoyancy refers to the ability of particles to float substantially free in solution that occurs when the density of the particle is substantially the same as the medium in which they are suspended. Coated particles formed in accordance with the present invention as described herein can reach buoyancy when there is approximately 30% contrast agent in the delivery medium, however, such levels can be adjusted for such preferred use according to techniques. described here.

One method of increasing the density of the particles is by using heavy water or deuterium oxide (D2O). When heavy water is used to swell the particles, the D2O displaces the H2O, which increases the weight of the particles for better levels of dispersion and buoyancy. Typically this leads to the possibility of adding greater amounts of contrast agent of at least about 5% using such a technique. However, some balancing effect can occur over time when the particles are brought into contact with an aqueous solution of contrast agent. Thus, it is preferred that when using D2O for this purpose, either suspension times are kept to a minimum or, more preferably, that the contrast agent is provided in a solution that also uses D2O.

Alternatively, the pH 1 particles can be neutralized with cesium hydroxide and / or the final neutralized particles can be equilibrated with cesium chloride. Such compounds diffuse cesium into the particles, such that either the cesium salt of polymethacrylic acid is formed or polymethacrylic acid is diffused and therefore enriched with cesium chloride.

Cesium increases the density of the particles, thus increasing the possibility of adding larger amounts of contrast agent. Typical levels of buoyancy can be adjusted using the cesium technique such that about 45 to about 50% contrast agent can be added to the delivery medium as desired for embolization. Cesium salts are non-toxic and make the particles visible using fluoroscopy. Cesium's atomic weight of 132.9 g / mol is slightly higher than that of iodine which provides beneficial effects including increasing overall density and enhancing X-ray contrast visibility, even without a contrast agent. For certain cancer treatments where a radioactive isotope of cesium is desired, such an active agent can be used as an alternative source of cesium by making the particles float in an embolic solution as well as being capable of being used as a source of active treatment.

The aforementioned techniques for improving the density of the particles, such as microparticles for embolization or other applications where density and / or buoyancy in solution are applicable properties that can be applied to the preferred particles described herein and / or can be apply for other similar particles. It should be understood that the disclosure is not limited to cesium and / or D2O treatment of the particles preferred herein and that such techniques may have broader implications on other particles such as other acrylic-based hydrogels and other polymeric particles.

As noted above, barium sulfate can be used between the core particles and the preferred or embedded PTFE coating within the core particles using any technique known or to develop in the art. Also, organic colorants can be similarly included in the core of the particles. These materials, particularly barium sulfate, also contribute to an increase in density, as well as provide radiopacity. In addition to a general increase in density as provided by the above D2O or cesium compounds, barium sulfate allows this benefit even after hydration.

ES 2 564 151 T3 substantial and / or total, allowing the suspended particles to remain isotonic. Thus, a barium sulfate powder coating can provide an inert precipitate that has no effect on physiological osmolarity.

It should be understood, based on this disclosure, that the various buoyancy additives mentioned above can be used independently or in combination to provide the most beneficial effects for a combination of core particles and the given coating.

The invention also includes particles for use in methods of delivering an active agent to a localized area on the body of a mammal. The method includes contacting the localized area with at least one of the particles of the invention as described above, such that an effective amount of the active agent is released locally to the area. Diseases or pathologies that can be treated by this method include any where localized or topical application of the active agent achieves some benefit in contrast to systemic absorption of the drug. Suitable active agents include NSAIDS, steroids, hormones, nucleic acids, agents used in the treatment of disorders of the gastrointestinal tract, such as ulcers, Crohn's disease, ulcerative colitis, and irritable bowel syndrome. Other active agents may include tacrolimus, sirolimus, paclitaxel, cis / carboplatin, antineoplastic agents, doxorubicin, and / or receptor blocking agents, eg, avp3 integrin blockers, that inhibit cell binding.

If the particle formulated for delivery of an active agent to a localized area is about 1 to about 1,000 pm in diameter, the drug-loaded microspheres can be applied to localized areas within the body of a mammal using syringes and / or catheters as a delivery device, without causing inadvertent occlusions. For example, using a contrast agent, a catheter can be inserted into the groin artery and its movement monitored until the area where localized administration is desired has been reached. A dispersion of the particles in a suitable injection medium can be injected through the catheter, ensuring only that a specific area of ​​the body will be treated with drug-loaded beads (particles), as will be understood by a person of With skill in the art, injection media include any pharmaceutically acceptable media that are known or developed in the art, such as, for example, saline, PBS or any other suitable physiological medium. According to a further embodiment described herein, the invention includes an injectable dispersion including particles and a contrast agent whose particles are substantially dispersed in solution. In a preferred embodiment, the particles are also detectable through fluoroscopy.

The polymeric particles of the invention can be used to prepare a sustained release formulation of an active agent for oral administration. The formulation comprises a particle, as described above, charged with an active agent. The polymeric particle used can be hollow, substantially hollow or solid. The particle can be loaded with the active agent either by dispersing or solvation of the active agent in the polymer solution prior to production of micro-sized particles through spray droplets, pelletizing a polymer melt, or performing a cryoextraction process. Alternatively, an uncharged polymer particle can be prepared and subsequently immersed in solutions containing active agents. The particles are then incubated in these solutions for a sufficient amount of time for the active agent to diffuse into the polymer matrix. After drying the particles, the active agent will be retained on the polymer particle. If this loading mechanism is used, drug loading can be controlled by adjusting the drug concentrations of the incubation medium and removing the particles from the incubation medium when an equilibrium condition has been reached.

Furthermore, it is contemplated that the active agent can be selected such that the action of the particles is complemented in a synergistic manner, especially if the particles are being used in an embolization or occlusive procedure. For example, if the tissue to which blood flow is desired to be minimized is tumor tissue, one may wish to load the particles used in the occlusion with a cytostatic drug, antiangiogenic agents, or an antimitotic drug.

Also provided is a method of tracking the passage of a particle through a blood vessel or other cavity in a mammalian body. The method includes injecting into the vessel, cavity, or a conduit adjacent to said cavity or vessel, at least one tracer particle, wherein the tracer particle is at least one particle prepared according to the procedures described above.

The tracer particle can include a contrast agent that can aid in visualization of the particle as it passes through the body cavity, blood vessels, and / or other locations. In general, smaller particles, such as those in the range of about 1 to about 10 pm, are preferred in this application, especially if the particles are to be injected into the blood stream. However, the particles can be of any size, as long as, for this purpose, they are not large enough to occlude the blood vessel, the body cavity, or the adjacent cavity or vessel to which the procedure is applied.

If the particles are loaded with a contrast agent, their movement can be visualized with X-ray machines, or any other contrast procedure, depending on the contrast agent used. However, if the

ES 2 564 151 T3 particles do not contain a contrast agent, the flow of the particles can be visualized using computed tomography based on ¹⁹F-NMR.

If desired, the tracer particle containing a contrast agent can be coated with a polymer coating. The polymer coating can comprise any polymer known or to be developed in the art, including any phosphazene polymers. If there is any toxicity or toxicity concern with respect to the contrast agent, it is desirable that the one or more of the coating agents be non-biodegradable. Depending on the nature of the visualization procedure, such contrast agents (for example, from the class of agents that enhance conventional radiographic contrast, such as ionic or non-ionic iodine-containing compounds (Imeron ™, Optiray ™, etc.) may be provided. .).

When using magnetic resonance imaging (MRI) for visualization, the contrast agent to be provided can be chosen from the class of rare earth compounds, such as Gadolinium and Samarium chelates, and so on. , as is well known in the art.

Since the hydrogel core component in embodiments of the present invention is derived from an anionic acrylate-based hydrogel polymer, such as polymethacrylic acid and the like, the incorporation of multivalent metal compounds, including the aforementioned rare earths or others metals, facilitates a favorable ionic interaction of these compounds, such as by ionic crosslinking or similar ionic interaction, thus providing favorable retention or accumulation of these compounds in the particles and therefore providing a sustained release effect of such compounds in various embodiments in accordance with the present invention.

The invention also includes particles for use in the method of performing an enhanced ultrasound imaging (sonography) procedure. In order to do this, at least one hollow microcapsule can be administered to the ultrasound subject to the area of ​​the ultrasound subject that is desired to be visualized. Such administration can be accomplished by any means known or to develop in the art, including through the use of a syringe, catheter, or other invasive or non-invasive medical device, and / or through a surgical incision. In such a method, it is preferable to use particles that are hollow or substantially hollow, that is, having an inner cavity that is equal to at least about 20%, at least about 30%, at least about 40%, at least about 50%. %, at least about 80%, at least about 90%, of the entire particle volume. The hollow particles are delivered to a portion of the ultrasound subject to be imaged. While not wishing to be bound by theory, it is speculated that the particles enhance the ultrasound image by increasing the ultrasound echo due to their abrupt density change, when compared to the surrounding tissue. The hollow cavities in the particles act to reflect the ultrasound, thereby enhancing the image.

Unless otherwise stated, temperatures are reported in degrees Celsius and pressure is atmospheric or near. An example of the preparation of a polyphosphazene of this invention is provided with the polymer synthesis of poly [bis (trifluoroethoxy) phosphazene] (Pzf), which can be prepared according to US Patent Application Publication No. 2003 / 0157142.

Also unless otherwise indicated, when a range of any type is disclosed or claimed, for example a range of molecular weights, layer thicknesses, concentrations, temperatures, and the like, it is intended to individually disclose or claim each possible number that such range could reasonably cover, including any sub-range covered therein. For example, when Applicants disclose or claim a chemical structural unit having a certain number of atoms, for example carbon atoms, Applicants' intention is to individually disclose or claim every possible number that such range could encompass, consistent with what is disclosed. here. Thus, by disclosing that an alkyl group or substituent may have 1 to 20 carbon atoms, Applicants' intention is to cite that the alkyl group has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 carbon atoms. In another example, by the disclosure that the microspheres have a diameter of about 500 to 600 pm, Applicants include within this disclosure the citation that the microspheres have a diameter of about 500 pm, about 510 pm, about 520 pm, about 530 pm, about 540 pm, about 550 pm, about 560 pm, about 570 pm, about 580 pm, about 590 pm, and / or about 600 pm, including any range or sub-range covered therein.

Example 1 (Reference)

Microparticle nuclei were formed in accordance with a preferred embodiment herein. A deionized water solution of polyvinyl alcohol (PVA) was prepared using about 23 g of PVA of weight average molecular weight of about 85,000-124,000, the PVA of which was about 87-89% hydrolyzed and 1000 g of water. A phosphate buffer solution was prepared using 900 g of deionized water, 4.53 g of sodium hydrogen phosphate, 0.26 g of sodium dihydrogen phosphate, and 0.056 g of ethylenediaminetetraacetic acid (EDTA). The methyl methacrylate (MMA) monomer was vacuum distilled prior to use.

ES 2 564 151 T3

The polymerization was carried out in a 2000-ml, round bottom, three-necked flask with an attached KPG mechanical stirrer. The flask was also equipped with a thermometer, reflux condenser, and a pressure release valve with a nitrogen inlet. The polymerization process used in addition 100 ml of the PVA solution prepared above, 900 ml of the phosphate buffer solution, 0.65 g of dilauroyl peroxide, 200.2 g of methacrylic acid methyl ester, and 2.86 g of triethylene glycol dimethacrylate. [00136] The pVa and buffer solutions were supplied to the reactor flask. The distilled MMA and triethylene glycol dimethacrylate were introduced, then dilauroyl peroxide was added to the same flask and the components were stirred to ensure dissolution of solids. The reaction flask was flushed with argon and the stirrer speed was set at 150 rpm to produce majority particle sizes in the 300-355mm range. Stirring continued for about 5 minutes. The stirrer was then set at 100 rpm and the argon wash was discontinued. The reaction flask was then subjected to a water bath which was heated to 70 ° C and held at approximately that temperature for approximately 2 hours. The bath temperature was then increased to 73 ° C and held for one hour, then the water bath temperature was raised again to 85 ° C and held for another hour. Stirring and heating were discontinued. The solution was filtered and the resulting polymethacrylate microparticle cores were dried in an oven at 70 ° C for approximately 12 hours. The microparticle cores were sieved and collected in size fractions of 100-150; 150-200; 200-250; 250-300; 300-355; 355-400; and 400 to 450 pm with a maximum performance at 300-355 pm.

The PMMA microparticle cores thus formed were then hydrolyzed. A 100 g portion of 250-300 pm size microparticle nuclei, 150 g of potassium hydroxide and 1,400 g of ethylene glycol were added to a 2000 ml flask, a reflux condenser with attached drying tube, and the mixture heated to 165 ° C for 8 hours for complete hydrolysis. The mixture was allowed to cool to room temperature, the solution was decanted, and the microparticle cores were washed with deionized water. The procedure was repeated for other calibrated sizes of microparticles (the following reaction times were applied: 300-355 micron particles: 10 hours, 355-400 micron particles: 12 hours and 400-455 micron particles: 14 hours). That is, the particular size of the particles can be selected, standardized, or calibrated according to the conditions under which they are prepared.

The microparticle cores were finally acidified with hydrochloric acid to a pH of 7.4, and dried in an oven at approximately 70 ° C.

Example 2 (Reference)

Microparticle nuclei formed according to Example 1 were then esterified in this Example. For the esterification surface treatment, 800 g of dry microparticle cores from Example 1 were weighed into a 2 L reaction vessel with a reflux condenser. 250 g of thionyl chloride in 1.5 L of diethyl ether were added with stirring. Stirring was continued at room temperature for 20 hours. The solvent and volatile reagents were removed by filtration and subsequently dried under vacuum. Then, 500 g of trifluoroethanol were introduced into 1.5 L of ether and the suspension was stirred for another 20 hours at room temperature. The particle cores were finally dried under vacuum.

Example 3 (Reference)

In an alternative surface treatment to Example 2, 800 g of dry microparticle cores from Example 1 were reacted with 1140 g of trifluoroethanol and 44 g of sulfuric acid was added as a catalyst. The mixture was stirred for 20 hours at room temperature, filtered and dried under vacuum.

Example 4 (Reference)

800 g of dry PMMA potassium salt microparticle cores that were partially esterified with trifluoroethanol as described above in Examples 1-2 were spray coated with PTFEP in an MP-I Precision Coater ™ fluidized bed coating apparatus ( available from Aeromatic-Fielder AG, Bubendor, Switzerland). The particle nuclei were collected by a stream of air (40-60 m³/ hr, 55 ° C inlet temperature) and coated with microdroplets of PTFEP solution from an air-fluid coaxial nozzle. The composition of the solution was 0.835 g of PTFEP, 550 g of ethyl acetate and 450 g of isopentyl acetate. A 10 to 30 g / min rate was fed through the 1.3 mm wide inner hole of the nozzle. At the head of the nozzle, it was atomized with pressurized air (2.5 bar). The total amount of the spray solution (3 kg) was calculated to cover the core of the particle with a 150 nm thick PTFEP film.

Example 5 (Reference)

The absorption of organic dyes was tested on microparticles formed according to Example 1. To 2 ml of phosphate buffered saline containing 1 ml of hydrated beads, a quantity of 5-10 µl of the respective dye was provided as a solution of 10 millimolar in ethanol. The samples were incubated for 30-60 minutes at room temperature with gentle shaking of the vial. The supernatant liquid was discarded and the particles were washed three times with 2 ml of either deionized water, saline, or PBS buffer prior to

ES 2 564 151 T3 visualization with light and fluorescence microscopy. The dyes tested included triphenylmethane-derived dyes such as fluorescein diacetate and Rhodamine 6G which were evaluated in conjunction with carbocyanine-based dyes such as Dil. The triphenylmethane-based fluorescein and Rhoamine dyes exhibited specific affinity for the hydrophilic PMMA hydrogel core through ionic interactions. They were able to easily withstand the harsh conditions of repeated washing and steam sterilization without substantial leaching. The carbocyanin dye Dil on the other hand exhibited high selectivity for the hydrophobic PTFEP layer, without penetrating the hydrophilic PMAA core material. Thus, with subsequent staining using the combination of Dil and fluorescein diacetate, both the core and the layer could be simultaneously visualized using a fluorescence light microscope. As a result, this procedure provides a fast, sensitive fluorescence staining assay for PMAA particles that simultaneously render the core and the shell visible under conditions encountered in real-world application. This procedure also allows the establishment of mechanical-elastic stress or damage to the PTFEP layer. The affinity of certain classes of dyes for the various components of the particle is further shown.

The use of these and other stains can be used to visually identify selected microspheres, which can be provided and stained for identification to indicate certain microsphere sizes for use in selected clinical or diagnostic applications. Color coding can also be used to identify microspheres selected on the basis of other properties, such as the content of certain therapeutic or diagnostic agents. Applications according to the present invention can also improve image display by enhancing the buoyancy behavior of the particles.

Figure 3A shows example microspheres A, B, and C of the present invention, in which the microspheres are each of different diameters, and each is color-coded differently. In an exemplary use of such microspheres of the present invention, color-coded microspheres of similar sizes can be packaged separately and supplied for use. Such color-coded microspheres can provide a user with a visual indication of specific microspheres in a particular clinical or diagnostic use.

In various embodiments in accordance with the present invention, the microspheres can be produced in calibrated sizes ranging from about 1 to about 10,000 nanometers in diameter. In one embodiment of the present invention, the microspheres of the present invention can be provided in sizes of about 40, about 100, about 250, about 400, about 500, about 700, and about 900 nanometers in diameter, with a visually distinctive color. imparted for each microsphere size. Other sizes, size ranges, and calibrated sized microspheres lacking color tint are also included in the present invention. Not only can microspheres or particles of different size ranges be provided, but their elasticity can be controlled in accordance with the present invention to specifically provide proximal or distal embolization behavior, due to potentially different ranges of compressibility that can alter the distance. displacement of the particles or microspheres after their release into a selected blood vessel. The microspheres of the present invention can also be provided in custom sizes and / or with custom colors as specified by a user for specific therapeutic or clinical diagnostic applications.

Example 6 (Reference)

Transarterial chemoembolization or TACE is a clinical procedure in which the blood supply to a tumor is interrupted by embolization and chemotherapy is delivered directly to the tumor. Selective embolization of tumor blood vessels without direct administration of chemotherapy (soft embolization) is also performed as a clinical procedure in certain situations.

In most living organisms with a developed circulatory system, the vascular system tends to shrink from the larger vessels proximal to the heart to the smaller vessels more distal to the heart. Thus, larger arteries tend to divide into smaller arteries, which eventually decreases the level of arterioles and the interface with small-diameter venules. Venous flow progresses from such venules through successively larger diameter veins as flow returns to the heart.

It is common, therefore, that blood vessels of different sizes can exist within a tumor mass or other target tissue. In a clinical situation, where embolization and maximum interruption of blood supply to a tumor or other target tissue is desired, serial embolization of progressively larger tumor vessels can provide more complete embolization, with or without delivery. chemotherapeutic or other therapeutic agents.

Figure 3B is a conceptual representation of a selective embolization of an exemplary artery 120 by serial administration of microspheres 121, 122, and 123 of different sizes. The direction of blood flow within exemplary artery 120 is shown by arrows in Figure 3B. In this example, microsphere 121 is the smallest diameter of the administered microspheres, and is injected first into artery 120, which occludes the vessel lumen at the diameter of the smallest vessel that will not allow microsphere 121 to pass. Continuing with this example, microsphere 122 is of intermediate diameter of the microspheres administered, and is injected first

ES 2 564 151 T3 in artery 120, which occludes the lumen of the vessel in the diameter of the smallest vessel that will not allow the passage of microsphere 122. Finally, in this example, microsphere 123 is the one with the largest diameter of the microspheres administered, and is injected first into artery 120, which occludes the vessel lumen at the diameter of the smallest vessel that will not allow the passage of microsphere 123. The result in this example is sequential blockage of blood flow at multiple levels throughout the blood supply of the tumor or target tissue.

In other examples of the present invention, fewer than three or more than three microspheres of different sizes can be administered to ensure the desired embolization of a tumor or other target tissue.

As provided in the previous examples of the present invention, the different sized microspheres of the present invention may further be provided with color coding to allow user identification and visual confirmation of sized microspheres in use at any stage of the procedure. clinical procedure.

The provision of microspheres of different sizes or other inherent qualities may be further facilitated by the use of shipping packaging and / or delivery devices that are color-coded to allow user identification and visual confirmation of the sized microspheres in use in any given stage of the clinical procedure in exemplary applications in accordance with the present invention. In various example applications of the present invention, such color-coded devices can be used in combination with the color-coding of the microspheres themselves, with the corresponding microsphere, and the color-coding of the packaging / delivery device.

Figure 3C shows a syringe used for packaging and / or delivery of color-coded microspheres of a selection size in accordance with the present invention. In the example shown in Figure 3C, the syringe 124 comprises a barrel 125, a plunger 126, a plunger tip 127, a Luhr-type injection tip 128, and a Luhr tip cap 129.

As shown in Figure 3C, one or more of the barrel 125 components, a plunger 126, a plunger tip 127, a Luhr-type injection tip 128, and a Luhr tip layer 129 may be colored in one color. common agreement with a color code to indicate a desired property of the microspheres contained therein. In an example of the present invention, a syringe may contain color-coded microspheres to indicate a certain size of the microspheres, and the syringe plunger, plunger tip, and Luhr tip layer may be similarly colored to further indicate the desired property of the contained microspheres to a user.