Cross-Reference to Related Application

None

Technical Field

The disclosed embodiments are in the field of producing biocompatible implants, and more particularly directed to a process for preparing an injectable biocompatible implant including a monodisperse suspension of polymer microspheres.

Background of the Art

Injectable biocompatible implants are well known in the art. These implants are used in a variety of cosmetic treatments including dermal filler applications, such as wrinkle or scar reduction; lifts to increase body volume; and others. U.S. Pat. No. 5,344,452 discloses a biocompatible implant including solid particles having smooth surfaces and having a diameter such that they cannot be metabolized, digested, absorbed or phagocytosed via lymph tracts or other tissue tracts from the implantation site. However, monodisperse populations of polymer microspheres are not taught.

Commercially available dermal fillers contain polymethylmethacrylate (PMMA) solid particles, such as Artefill by Artes Medical, Inc. of San Diego, CA, US. However, the particles are not of uniform size, and the product contains an amount of solid particles having a diameter of less than 5 μιη. Particles of 5 μιη or less are prone to macrophagosis and migration into the circulatory system. Studies have concluded that there is a specific threshold of particle size that is critical to avoid phagocytosis by macrophages and giant cell formation with resulting granulomatous inflammation. Associated observations suggest that small PMMA particles, less than 20 μιη in diameter, engender a foreign body response.

Polymer Microsphere Synthesis

Mechanically ground polymer resin or pellets of 20 μιη to 50 μιη size are unsuitable for dermal filler applications due to sharp edges or points on the particles which may result in injury or inflammation of the treated area. Methods of choice for synthesis of polymer particles with spherical shape and smooth surfaces include emulsion polymerization, seeded polymerization, dispersion polymerization, and suspension polymerization. Each type of polymerization, however has its limitations.

Emulsion polymerization can allow synthesis of very uniform particles with a coefficient of variation of less than 5 %. However, the maximum particle size that can be prepared is about 2 μιη.

Dispersion polymerization is another technique for synthesis of uniform and spherical polymer microspheres. A limitation of this technique is that it is very difficult to incorporate a crosslinker, such as a diacrylate, in the polymerization process without impacting the polymer yield. Crosslinking is beneficial to the physical strength of the microspheres. In the present process, crosslinking is also beneficial for the elevated temperature cleaning step.

Some prior art dermal fillers are comprised primarily of collagen or hyaluronic acid. These types of dermal filler have a short effective duration due to absorption or metabolism of the filler by the host patient. Typical duration is from three months to two years, depending on the molecular weight of the filler. After this time repeat injections are necessary to extend the effects of the treatment. In addition, the commonly used bovine collagen necessitates skin testing prior to injection to reduce the incidence of hypersensitivity. Although the majority of side effects caused by hypersensitivity are mild and transient, rare cases of granuloma and inflamed nodule formation have been reported.

There remains a need for an injectable biocompatible implant in which polymer particles with novel and advantageous physical properties are administered to provide enduring and benign implant treatments.

Summary

Shortcomings of the prior art injectable implants are remedied at least in part by a method for preparing an injectable biocompatible implant comprising the following steps:

(a) forming a microsphere suspension by means of seeded polymerization, said

microsphere suspension including:

(i) a multiplicity of polymer microspheres;

(ii) at least one liquid selected from the group consisting of water and organic solvents; and,

(iii) residual chemicals;

(b) mechanically separating from said microsphere suspension said polymer

microspheres having a diameter of less than about 20 μιη;

(c) measuring the amount of said polymer microspheres having a diameter of less than about 20 μιη and comparing said amount to a predetermined level, and:

(i) if said amount is greater than said predetermined level, returning to (b); and, (ii) if said amount is less than or equal to said predetermined level, said microsphere suspension being a monodisperse microsphere suspension wherein:

(A) said polymer microspheres of said monodisperse microsphere

suspension have an average diameter of between about 20 μιη and about 50 μιη; and,

(B) the diameters of said polymer microspheres of said monodisperse microsphere suspension have a coefficient of variation of less than 10%;

(d) elevated temperature cleaning said monodisperse microsphere suspension so that said residual chemicals are reduced to a predetermined level, forming a microsphere preparation; and,

(e) combining said microsphere preparation with a suspension agent so that said

microsphere preparation is substantially evenly distributed within said suspension agent, forming said injectable biocompatible implant.

In one embodiment, the polymer microspheres are made of polymethylmethacrylate.

In another embodiment, diameters of microspheres in the monodisperse microsphere suspension have a coefficient of variation of less than 8%.

In another embodiment, the suspension agent is a collagen of porcine origin. Further provided are injectable biocompatible implants produced according to these processes.

Other embodiments, in addition to the embodiments enumerated above, will become apparent from the following detailed description, taken in conjunction with the accompanying drawings, which illustrate, by way of example, the principles of the process for preparing an injectable biocompatible implant and product thereof.

Brief Description of the Drawings

The inventive aspects of the following disclosure will be best understood when reference is made to the appended drawings and the detailed description thereof, in which identical parts are identified by identical reference numbers and wherein:

FIG. 1 is a flow chart of a process for preparing an injectable biocompatible implant;

FIG. 2 is a graph of a prior art particle size distribution of suspension polymerized solid particles;

FIG. 3 are graphs of particle size distributions at various steps of the present process: A) after forming a microsphere suspension, B) after mechanically separating small microspheres from the microsphere suspension, and C) after forming a monodisperse microsphere suspension;

FIG. 4 is a line drawing of a Scanning Electron Microscopy (SEM) image of the microsphere preparation showing monodispersity of the collection of microspheres;

FIG. 5 is a line drawing of an SEM image of the microsphere preparation showing substantial sphericity of the microspheres;

FIG. 6 is a flow chart of a process for preparing an injectable biocompatible implant including an anesthetic;

FIG. 7 is a flow chart of a process for preparing an injectable biocompatible implant including two microsphere preparations;

FIG. 8 is the SEM image upon which the line drawing of FIG. 4 is based; and,

FIG. 9 is the SEM image upon which the line drawing of FIG. 5 is based. Detailed Description of the Invention

A process is provided for preparing an injectable biocompatible implant. The implant includes polymer microspheres produced by seeded polymerization. Seeded polymerization techniques produce particles with high sphericity and a narrow particle size distribution. The present process provides a method for treating a microsphere suspension to remove microspheres having a diameter of less than about 20 μιη, and further cleaning the suspension to remove residual chemicals from the polymerization process.

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 interpretation of the claims and the specification.

A biocompatible implant is a material for implantation in the body with the ability to perform its desired function with respect to a medical therapy without eliciting any undesirable local or systemic effects in the host.

The term micron and abbreviation 'μιη' both mean micrometer.

Micron sized means having a diameter on the order of microns.

A microsphere or microspheres means a single micron sized particle, or collection of particles, having a highly spherical shape. A collection of microspheres is in general highly spherical, although some individual particles within the collection may have lower sphericity.

Seeded polymerization refers to a family of polymerization techniques capable of producing uniform micron sized polymer particles. Seeded polymerization processes begin with a collection of sub-micron sized polymer particles, referred to as seeds. The seeds are treated in one or more swelling steps, resulting in the micron sized polymer particles. The seeded polymerization process may be repeated multiple times to ensure that the seeds grow to a target diameter. For example, a target diameter of 2 μιη to 5 μιη may be achieved from submicron seeds in 2 to 5 seeded polymerization steps. A target diameter of 35 μιη may be reached using additional seeded polymerization steps wherein the seeds arelO μιη to 20 μιη particles, themselves prepared from smaller seeds. Examples of seeded polymerization techniques include seeded emulsion polymerization, seeded dispersion polymerization, seeded suspension polymerization, and others well known in the art.

Residual chemicals refers to a group of components of a polymerization process which remain in a polymer suspension after completion of the process. The group includes unreacted monomer, swelling agent, dispersant, surfactant, and any other components of the process.

Mechanically separating means dividing a mixture into two or more constituent parts by mechanical means. Such means include filtration, centrifugation, gravity separation, and others well known in the art. The term as used herein encompasses a combination of two or more mechanical separation techniques.

Monodisperse, as used herein to apply to a collection of particles, means that particles within the collection have substantially the same size. The degree of monodispersity of a collection is quantified by the coefficient of variation, in this case variation of the diameter of particles in the collection.

Elevated temperature cleaning means a process for separating residual chemicals from a polymer suspension, performed at temperatures above room temperature. Such processes include steam stripping, reflux, and others well known in the art.

Substantially evenly distributed means an evenness of distribution adequate for smooth injection.

Diacrylate and triacrylate mean compounds having two or three acrylate groups, respectively.

Collagens of human origin include autologous collagen, cadaver-derived collagen, human-bioengineered collagen, and others well known in the art.

Referring initially to FIG. 1, a process is provided for preparing an injectable biocompatible implant, comprising:

(a) forming a microsphere suspension by means of seeded polymerization, said microsphere suspension including:

(i) a multiplicity of polymer microspheres;

(ii) at least one liquid selected from the group consisting of water and organic solvents; and,

(iii) residual chemicals; (b) mechanically separating from said microsphere suspension said polymer microspheres having a diameter of less than about 20μιη;

(c) measuring the amount of said polymer microspheres having a diameter of less than about 20 μιη and comparing said amount to a predetermined level, and:

(i) if said amount is greater than said predetermined level, returning to (b); and,

(ii) if said amount is less than or equal to said predetermined level, said microsphere suspension being a monodisperse microsphere suspension wherein:

(A) said polymer microspheres of said monodisperse microsphere suspension have an average diameter of between about 20 μιη and about 50 μιη; and,

(B) the diameters of said polymer microspheres of said monodisperse microsphere suspension have a coefficient of variation of less than 10%;

(d) elevated temperature cleaning said monodisperse microsphere suspension so that said residual chemicals are reduced to a predetermined level, forming a microsphere preparation; and,

(e) combining said microsphere preparation with a suspension agent so that said microsphere preparation is substantially evenly distributed within said suspension agent, forming said injectable biocompatible implant.

FIG. 2 is a graph of a prior art particle size distribution of suspension polymerized PMMA microspheres. The typical particle size distribution of suspension polymerized PMMA solid particles follows a Gaussian, or bell-shaped, distribution. While the shown distribution is centered at 40 μιη, a significant percentage of particles in the population are outside of the desired size range of about 20 μιη to about 50 μιη.

The preferred seeded polymerization technique of step (a) is seeded emulsion polymerization. FIGS. 3A-3C are graphs of particle size distributions at various steps of the present process. FIG 3A is a graph of the particle size distribution after forming a microsphere suspension by means of seeded emulsion polymerization in step (a). The main peak of 35 μιη diameter is the result of the step- wise seeded emulsion polymerization. The diameter of this main peak can be selected by calculating the amount of monomer needed to swell the preexisting monosized seeds to the target size of between about 20 μιη to about 50 μιη.

Terms referring to the diameter of the particle size distribution, such as 'about 20 μιη' , are intended to encompass a population with an average diameter of the value stated and a coefficient of variation (CV) low enough that the population is considered monodisperse. For example, a population with a 20 μιη average diameter and 10 % CV will contain some particles with diameters under 20 μιη. Such a population is encompassed in the term 'about 20 μιη'.

As shown in FIG. 3A, the seeded emulsion polymerization technique produces a subset of particles with an average diameter of less than one micron to several microns, referred to below as small particles. The presence of these small particles is due to the homogeneous nucleation mechanism which causes competition for monomer at two polymerization sites: the seed particles themselves and the water in which the seed particles are dissolved. Formation of these small particles can be suppressed by adding water soluble polymerization inhibitor, but such formation cannot be eliminated entirely. Particles in this size range are hazardous to the human body due to the potential for migration and macrophagosis.

FIG. 3B is a graph of the particle size distribution after mechanically separating small particles from the microsphere suspension in step (b). The population of undesirable small particles is significantly reduced by the mechanical separation step. However, it has not yet been reduced to the level predetermined to be necessary for this application.

FIG. 3C is a graph of the particle size distribution after forming a monodisperse microsphere suspension in step (c). In the shown example, after several repetitions of the mechanical separation step (b), the level of particles smaller than 20 μιη reached 0.05 wt %. The necessary level of reduction of particles having a diameter of less than about 20 μιη must be predetermined based on the requirements of the application. Using the present process it is possible to completely or nearly completely remove microspheres smaller than about 20 μιη.

FIG. 4 is a line drawing of a Scanning Electron Microscopy (SEM) image of the microsphere preparation showing monodispersity of the collection of microspheres. In the imaged field of approximately 500 microspheres, no microspheres of less than 5 μιη diameter are detectable. FIG. 8 is the SEM image upon which the line drawing of FIG. 4 is based.

FIG. 5 is a line drawing of an SEM image of the microsphere preparation showing substantial sphericity of the microspheres. Low surface roughness of the microspheres is also apparent in the image. Due to the surface tension of the monomer- swollen seed particles prior to and during the polymerization, microsphere particles have very high sphericity when the polymerization process is complete. High sphericity and low surface roughness improve injectability while reducing the potential for deleterious effects at the injection site. FIG. 9 is the SEM image upon which the line drawing of FIG. 5 is based.

In step (d), residual chemicals are reduced to a predetermined biocompatible level based upon the requirements of the application. In seeded polymerization, the amount of unreacted monomer can be as high as 1 wt % of the final suspension and must be reduced or eliminated before the suspension can safely be used for implant purposes. In a preferred embodiment, the elevated temperature cleaning of (d) reduces residual chemicals to less than about 100 parts per million of the microsphere suspension.

The combined microsphere preparation and suspension agent of step (e) should be sterilized prior to injection in keeping with accepted medical practice.

In an embodiment, the polymer of the polymer microspheres of (a) is

polymethylmethacrylate.

In another embodiment, the polymer of the polymer microspheres of (a) is

polymethylmethacrylate crosslinked with a compound selected from the group consisting of diacrylates and triacrylates. PMMA crosslinking increases microsphere strength and solvent resistance.

In another embodiment, the polymer microspheres of the monodisperse microsphere suspension of step (c) have an average diameter of between about 30 μιη and about 50 μιη.

In another embodiment, diameters of the polymer microspheres of the monodisperse microsphere suspension of step (c) have a coefficient of variation of less than 8%.

In another embodiment, the suspension agent of step (e) is a collagen. In preferred embodiments, the collagen is of porcine origin, bovine origin, or human origin. Because of the greater biological similarity between porcine and human skin versus bovine and human skin, porcine collagen suspension agents have potentially lower immunogenicity than bovine collagen suspension agents. In addition, porcine collagen does not require pre-procedure sensitivity testing and eliminates the possibility of patients being infected by the contagious mad cow disease.

In another embodiment, the suspension agent of step (e) is hyaluronic acid. Commercially available injectable hyaluronic acid is virtually identical to that produced by the human body; therefore allergenic testing is usually not required for its use. In another embodiment, the suspension agent of step (e) is selected from the group consisting of a biocompatible synthetic, a natural polymer, and a polymer blend. In preferred embodiments, the suspension agent is carboxymethyl cellulose or hydroxypropyl

methylcellulose.

FIG. 6 is a flow chart of a process for preparing an injectable biocompatible implant further including combining said injectable biocompatible implant of step (e) with up to 1% by weight of a local anesthetic. In a preferred embodiment, the local anesthetic is lidocaine.

FIG. 7 is a flow chart of a process for preparing an injectable biocompatible implant further including:

the microsphere preparation of step (d) being a first microsphere preparation; the first microsphere preparation having a first average diameter; forming a second microsphere preparation by duplicating step (a) through step (d), said microsphere preparation of step (d), as duplicated, being a second microsphere preparation;

the second microsphere preparation having a second average diameter;

the first average diameter and the second average diameter differing by at least about 10%; and,

in (e), the microsphere preparation being the first microsphere preparation, further combining the second microsphere preparation with the suspension agent so that the second microsphere preparation is substantially evenly distributed within the suspension agent.

Because the CV of microsphere preparations produced according to the present process is so narrow, often in the range of 2-8 %, there is an advantage in mechanical separation (b) and cleaning (d) steps, which makes the process possible. However, it was found experimentally that a mixture of two microsphere preparations having different average diameters increased ease of injecting the final implant. Further provided is a mixture of more than two microsphere preparations having different average diameters, for example four preparations having average diameters of 25 μιη, 30 μιη, 35 μιη, and 40 μιη.

Further provided are the products of these processes and their preferred embodiments, either alone or in combination.

Further provided is a kit, including an injectable biocompatible implant prepared by the present process, and a syringe filled with about 1 mL to about 3 mL of the implant. The contents of the kit are sterilized and packaged for ready use. The embodiments of the process and product described herein are exemplary and numerous modifications, combinations, variations, and rearrangements can be readily envisioned to achieve an equivalent result, all of which are intended to be embraced within the scope of the appended claims. Further, nothing in the above-provided discussions of the process and product should be construed as limiting the invention to a particular embodiment or combination of embodiments.

Examples

The present process and product thereof are further defined in the following Examples. These Examples are given by way of illustration only, and should not be construed as limiting the invention to a particular embodiment or combination of embodiments. Numerous modifications, combinations, variations, and rearrangements can be readily envisioned to achieve an equivalent result or adapt the process to various uses and conditions, all of which are intended to be embraced within the scope of the appended claims. The scope of the invention is defined by the appended claims.

The meaning of abbreviations used is as follows: "g" means gram(s), "mg" means milligram(s), "μΕ" means microliter(s), "mL" means milliliter(s), "min" means minute(s), "h" means hour(s), "vol %" means percent by volume, "wt %" means percent by weight, "μιη" means micrometer(s), and "rpm" means revolutions per minute.

Example 1: Preparation of injectable biocompatible implant and testing

on laboratory mice

A PMMA microsphere suspension was obtained from Magsphere, Inc. of Pasadena, CA, US. The suspension was formed by means of seeded polymerization and contained crosslinked PMMA microspheres. 50 g of the microsphere suspension was dispersed in 500 mL of distilled water. The mixture was mechanically stirred in a container for 30 min. The stirring was stopped and the mixture was allowed to rest for 1 h, during which particles larger than about 20 μιη settled to the bottom of the container. At the end of 1 h, the top layer, which contained fine particles, was decanted. The procedure of stirring and settling was repeated 10 times to ensure that the fine particle concentration was reduced to a predetermined level. The low level of fine particles was generally indicated by the clear supernatant of the microsphere suspension.

The supernatant was drained, and PMMA microspheres, with fine particles removed, were then combined with 70 vol % isopropyl alcohol in a 1 : 10 volume ratio. The mixture was stirred for 5 h. After stirring, the PMMA microspheres were allowed to settle and the supernatant removed. The process of combining with solvent, stirring, and settling was repeated 5 times. The result of this process was a monodisperse microsphere suspension.

The monodisperse microsphere suspension was transferred to a two-part resin reactor for reflux. Elevated temperature cleaning of the monodisperse microsphere suspension was carried out by means of reflux in 500 mL of 90 vol % isopropyl alcohol for 18 h. The cleaning step dissolved most of the remaining soluble species in the suspension in the isopropyl alcohol solvent. Due to the microspheres being crosslinked, elevated temperature cleaning does not reduce the integrity of the microspheres. A microsphere preparation was formed by the cleaning step.

The microsphere preparation was rinsed with 70 vol % isopropyl alcohol and dried, forming a microsphere powder suitable for further formulation as an injectable biocompatible implant.

FIG. 4 is a Scanning Electron Microscopy (SEM) image of the microsphere preparation showing monodispersity of the collection of microspheres. In the imaged field of approximately 500 microspheres, no microspheres of less than 5 μιη diameter are detectable.

FIG. 5 is an SEM image of the microsphere preparation showing substantial sphericity of the PMMA microspheres. Low surface roughness of the microspheres is also apparent in the image. The amount of residual chemicals in the final isopropyl alcohol rinse after the cleaning step was determined by evaporating a known amount of the rinse alcohol and measuring the residual weight. The total residual was found to be less than 100 parts per million in the final rinse.

An injectable biocompatible implant was formed by combining 10 wt % PMMA microsphere preparation and 100 mL of saline. The saline was the suspension agent in this implant. A 100 mL implant was subcutaneously injected into each of eight albino inbred mice. The group of mice consisted of 4 males and 4 females. All mice were eight weeks of age. The mice were observed for two months, after which there was no detectable acute toxicity.

Example 2: Preparation of monodisperse microsphere suspension by

mechanical filtration

A PMMA microsphere suspension was obtained from Magsphere, Inc. of Pasadena, CA, US. 50 g of the microsphere suspension was suspended in 500 mL of distilled water. The mixture was filtered through a screen with 15 μιη opening size to remove unwanted fine PMMA particles. The retained PMMA microspheres were resuspended in 500 mL of distilled water and the procedure of filtration and resuspension was repeated at least 10 times to ensure that the fine particle concentration was reduced to a predetermined level. This procedure resulted in a monodisperse microsphere suspension.

Example 3: Preparation of monodisperse microsphere suspension by light centrifugation

A PMMA microsphere suspension was obtained from Magsphere, Inc. of Pasadena, CA, US. 50 g of the microsphere suspension was mixed with 450 mL of distilled water. The mixture was centrifuged at 500 rpm for 10 min. The supernatant, which contained mostly submicron sized PMMA particles, was decanted. This process was repeated 10 times or until submicron sized particles in the mixture were reduced to a predetermined level. This procedure resulted in a monodisperse microsphere suspension. Example 4: Preparation of injectable biocompatible implant with porcine collagen suspension agent

A PMMA microsphere preparation suitable for further formulation as an injectable biocompatible implant was prepared by the method described in Example 1. The microsphere preparation was monodisperse, with an average diameter of 40 μιη. The injectable biocompatible implant was formed by mixing: 400 mg of the microsphere preparation, 28 mg of lyophilized porcine powder dissolved in 2 mL of distilled water, and 0.3 wt % lidocaine. This mixture formed a stable suspension which, after sterilization, was injectable by a 25 gauge hypodermic needle.

Example 5: Preparation of injectable biocompatible implant with

hyaluronic acid suspension agent

A PMMA microsphere preparation suitable for further formulation as an injectable biocompatible implant was prepared by the method described in Example 1. The microsphere preparation was monodisperse, with an average diameter of 40 μιη . The injectable

biocompatible implant was formed by mixing: 400 mg of the microsphere preparation, 34.4 mg of hyaluronic acid dissolved in 2 mL of phosphate buffer, and 0.3 wt % lidocaine. This mixture formed a stable suspension; no settling of the microspheres was observed after 48 h at room temperature. The implant was injectable by a 25 gauge hypodermic needle.

Example 6: Preparation of injectable biocompatible implant with sodium carboxymethyl cellulose suspension agent

A PMMA microsphere preparation suitable for further formulation as an injectable biocompatible implant was prepared by the method described in Example 1. The microsphere preparation was monodisperse, with an average diameter of 30 μιη. The implant was formed by mixing: 400 mg of the microsphere preparation and 2 mL of 2 wt % sodium carboxymethyl cellulose in water. This mixture formed a stable, injectable suspension. Example 7: Preparation of injectable biocompatible implant as a mixture of two monodisperse microsphere suspensions having different average

diameters

Two PMMA microsphere preparations suitable for further formulation as injectable biocompatible implants were prepared by the method described in Example 1. Both microsphere preparations were monodisperse, the first preparation having an average diameter of 30 μιη and the second preparation having an average diameter of 40 μιη. The implant was formed by mixing: 200 mg of the microsphere preparation with 30 μιη average diameter, 200 mg of the microsphere preparation with 40 μιη average diameter, and 35 mg of hyaluronic acid dissolved in 2 mL of phosphate buffer. This mixture formed a stable suspension, and was injectable by a 25 gauge hypodermic needle.

While monodispersity of the microsphere suspension allows mechanical separation of unwanted submicron sized particles, it was found that a mixture of two or more monodisperse microsphere suspensions having different average diameters was advantageous in the final injectable biocompatible implant.