Link:  Pharm/Biotech Resources

Title:  Slow release protein polymers

United States Patent:  6,939,557

Issued:  September 6, 2005

Inventors:  Rowe; Stephen C. (Wellesley, MA); Yim; Kalvin (North Andover, MA); Retnarajan; Beadle P. (Beverly, MA); Hubbell; Jeffrey A. (Zumikon, CH); Annavajula; Durga (Acton, MA)

Assignee:  Azopax Therapeutics LLC (Wellesley, MA)

Appl. No.:  650115

Filed:  August 26, 2003

The invention features articles for delivery of a biologically active substance, methods for making such articles, and methods for treating an animal using the articles.

SUMMARY OF THE INVENTION

The present invention features articles for delivery of a biologically active substance (hereafter "BAS"), and methods for making such articles. The articles of the invention improve the bioavailability of the BAS by formulating the BAS in an insoluble form. The invention also features methods of treating an animal using the articles for delivery of a BAS.

Accordingly, in a first aspect the invention features a biocompatible therapeutic article for delivery of a BAS, comprising a macromer, a molecule or mixture of molecules which preferentially excludes proteins, and the BAS, wherein the BAS is in an insoluble format upon completion of the formulation of the article comprising the macromer, molecule, or mixture of molecules which preferentially excludes proteins, and BAS.

In a preferred embodiment of the first aspect of the invention, the biocompatible therapeutic article has at least one of the following properties: the BAS is less than 15% aggregated; the article contains at least 10% macromer and at least 5% BAS, as measured by dry weight; the time at which 5% of the releasable BAS is released from the article is greater than 1/16 of t₅₀; or the t₅₀ is greater than or equal to ⅝ of t₈₀. More preferably the biocompatible therapeutic article has at least two of the above properties. Most preferably, the biocompatible therapeutic article has all of the above properties.

In another embodiment of the first aspect of the invention, the molecule which preferentially excludes proteins is a macromer, poly(ethylene glycol), hyaluronic acid, or poly(vinylpyrrolidone). In yet another embodiment, the macromer is a hydrogel. In still another embodiment, the solubility of a protein in the article comprising the macromer, molecule that preferentially excludes proteins, and BAS is less than 5-10 mg/ml, and more preferably is less than 1 mg/ml.

In another embodiment of the first aspect of the invention, the mixture of molecules comprises a positively charged ion-carrying reagent, for example, triethanolamine or Tris, when the pH is such that the protein is negatively charged. In still another embodiment, the mixture of molecules comprises a negatively charged ion-carrying reagent, such as sodium dodecyl sulfate, when the pH is such that the protein is positively charged. In yet another embodiment, the mixture of molecules comprises a surfactant, for example, Tween 20, Tween 80, or poloxamer F68. In a second aspect, the invention features a method for making a therapeutic article for delivery of a BAS, involving (a) combining the BAS with a molecule or mixture of molecules which preferentially excludes proteins; (b) combining the mixture formed in step (a) with a macromer, wherein the BAS is in an insoluble form and remains insoluble upon combining with the molecule or mixture of molecules which preferentially excludes proteins and the macromer; (c) forming a mixture of the combination formed in step (b); and (d) polymerizing the mixture to form an article.

In one embodiment of the second aspect of the invention, steps (a) and (b) are combined into a single combination step.

In a preferred embodiment of the second aspect of the invention, the biocompatible therapeutic article has at least one of the following properties: the BAS is less than 15% aggregated; the article contains at least 10% macromer and at least 5% BAS, as measured by dry weight; the time at which 5% of the releasable BAS is released from the article is greater than 1/16 of t₅₀; or the t₅₀ is greater than or equal to ⅝ of t₈₀. More preferably the biocompatible therapeutic article has at least two of the above properties. Most preferably, the biocompatible therapeutic article has all of the above properties.

In another embodiment of the second aspect of the invention, the molecule which preferentially excludes proteins is a macromer, poly(ethylene glycol), hyaluronic acid, or poly(vinylpyrrolidone). In yet another embodiment, the macromer is a hydrogel. In yet another embodiment, the macromer is a hydrogel. In still another embodiment, the solubility of a protein in the article comprising the macromer, molecule that preferentially excludes proteins, and BAS is less than 5-10 mg/ml, and more preferably is less than 1 mg/ml.

In another embodiment of the second aspect of the invention, the mixture of molecules comprises a positively charged ion-carrying reagent, for example, triethanolamine, when the pH is such that the protein is negatively charged. In still another embodiment, the mixture of molecules comprises a negatively charged ion-carrying reagent, such as sodium dodecyl sulfate, when the pH is such that the protein is positively charged. In yet another embodiment, the mixture comprises a surfactant, for example, Tween 20, Tween 80, or poloxamer F68.

In a third aspect the invention features a method of treating an animal, involving administering the biocompatible therapeutic article of the first aspect of the invention to a mammal. Preferably the mammal is a rodent, and most preferably the mammal is a human.

In yet other preferred embodiments, the articles are administered to the lung of the mammal, or are administered intravenously, subcutaneously, intramuscularly, orally, or nasally.

In a preferred embodiment of any of the above aspects of the invention, the macromer comprises: (a) a region forming a central core; (b) at least two degradable regions attached to the core; and (c) at least two polymerizable end groups, where the polymerizable end groups are attached to the degradable regions. In preferred embodiments, the region forming a central core is a water soluble region. The water soluble region may be poly(ethylene glycol), poly(ethylene oxide), poly(vinyl alcohol), poly(vinylpyrrolidone), poly(ethyloxazoline), poly(ethylene oxide)-co-poly(propylene oxide) block copolymers, polysaccharides, carbohydrates, proteins, and combinations thereof. The degradable region is selected from the group consisting of poly(α-hydroxy acids), poly(lactones), poly(amino acids), poly(anhydrides), poly(orthoesters), poly(orthocarbonates), and poly(phosphoesters). Preferably, the poly(α-hydroxy acid) is poly(glycolic acid), poly(DL-lactic acid), or poly(L-lactic acid), and the poly(lactone) is poly(ε-caprolactone), poly(δ-valerolactone), or poly(γ-butyrolactone). In another preferred embodiment, the degradable region comprises poly(caprolactone). In yet another embodiment, the polymerizable end groups contain a carbon-carbon double bond capable of polymerizing the macromer.

In other embodiments of the above aspects of the invention, the macromer includes: (a) a water soluble region comprising a three-armed poly(ethylene glycol) with a molecular weight of 3,000 to 6,000 daltons; (b) lactate groups attached to the region in (a); and (c) acrylate groups capping the region in (b). The macromer may alternatively include: (a) a water soluble region comprising poly(ethylene glycol) with a molecular weight of either 2,000 or 3,400 daltons; (b) lactate groups on either side of the region in (a); and (c) acrylate groups capping either side of the region in (b). In another alternative, the macromer may include (a) a water soluble region comprising poly(ethylene glycol) with a molecular weight of 3,400 daltons; (b) caprolactone groups on either side of region in (a); and (c) acrylate groups capping either side of the region in (b).

In still other embodiments of any of the above aspects of the invention, the article includes at least 5%, more preferably 10%, and most preferably 20-30% active substance by dry weight. In still another embodiment, the article is biodegradable.

In a more preferred embodiment of any of the above aspects of the invention, the macromer includes a water soluble region consisting of a three-armed PEG with a molecular weight of 4,200 to 5,400 daltons; lactate groups one end of each arm of the PEG; and acrylate groups capping the lactate groups.

In another more preferred embodiment of the above aspects of the invention, the macromer is made of a triad ABA block copolymer of acrylate-poly(lactic acid)-PEG-acrylate-poly(lactic acid)-acrylate. The PEG has a MW of 3,400 daltons; the poly(lactic acids) on both sides had an average of about five lactate units per side; and the macromer is therefore referred to herein as "3.4kL5." In another more preferred embodiment, a lower molecular weight PEG, such as MW 2,000 daltons PEG is used in place of the MW 3,400 PEG, and the resulting macromer is abbreviated as "2kL5."

In yet another more preferred embodiment of the above aspects of then invention, the macromer is an acrylate-PCL-PEG-PCL-acrylate macromer. The PEG has a MW of 3,400 daltons and has polycaprolactone on both sides, with an average of about 6 caproyl units per side. This macromer is referred to herein as "3.4kC6."

In other preferred embodiments, the BAS is a protein or peptide. More preferably the protein is chosen from a group consisting of hormones, antibodies, differentiation factors, angiogenic factors, enzymes, cytokines, chemokines, interferons, colony-stimulating factors, and growth factors. Most preferably, the protein is a hormone, such as human growth hormone, or a peptide, such as LHRH.

In still other embodiments of the second and third aspects of the invention, the therapeutic articles release at least 80% of the BAS at a time 1¼ times greater than t₅₀. At least 80% of the therapeutic articles may have a particle size of less than about 80 microns. The water soluble region may consist essentially of PEG having a molecular weight of about 500 to 20,000 daltons, and more preferably, between 1,000 and 10,000 daltons. The degradable region may comprise a blend of at least two different polymers. In addition, the macromer may be non-degradable.

In still other embodiments of the second and third aspects of the invention, the therapeutic article is capable of releasing the BAS for at for a period of time at least 2 times greater than t₅₀. The article is also capable of delivering a therapeutic dose of the BAS for at for a period of time at least 1¼ times greater than t₅₀.

DETAILED DESCRIPTION OF THE INVENTION

The invention provides methods and compositions for the administration of a biologically active substance (BAS) in an insoluble format. The compositions of the invention improve the bioavailability of the BAS by formulating the BAS in an insoluble format. These methods and compositions provide for the controlled, sustained delivery of relatively large quantities of these substances, with a low burst effect.

Macromers

The macromers of the present invention have at least one region forming a central core, at least one degradable (e.g., hydrolyzable) region, and at least one polymerizable region. The macromers may be water-soluble or water insoluble. Preferably, the region forming a central core is water soluble. If desired, the macromers may be polymerized to form hydrogels, which are useful for delivering incorporated substances at a controlled rate. Methods to formulate macromers and shape them into articles are described, for example in WO 99/03454, hereby incorporated by reference. An important aspect of the macromers is that the polymerizable regions are separated by at least one degradable region. This separation facilitates uniform degradation in vivo.

The ratio between the central core region and the hydrolyzable region of the macromer determines many of the general properties of the macromer. For example, the water solubility of the macromers can be controlled by varying the percentage of the macromer that consists of hydrophobic degradable groups.

There are several variations of the macromers of the present invention. For example, the polymerizable regions can be attached directly to the degradable regions; alternatively, they can be attached indirectly via water-soluble, nondegradable regions, with the polymerizable regions separated by a degradable region. For example, if the macromer contains a single water-soluble region coupled to a degradable region, one polymerizable region can be attached to the water-soluble region, and the other to the degradable region.

In another embodiment, a water-soluble region forms the central core of the macromer and has at least two degradable regions attached to it. At least two polymerizable regions are attached to the degradable regions so that, upon degradation, the polymerizable regions, particularly in the polymerized gel form, are separated. Alternatively, if the central core of the macromer is formed by a degradable region, at least two water soluble regions can be attached to the core, and polymerizable regions are attached to each water soluble region.

In still another embodiment, the macromer has a water-soluble backbone region, with a degradable region attached to the macromer backbone. At least two polymerizable regions are attached to the degradable regions, such that they are separated upon degradation, resulting in gel product dissolution. In a further embodiment, the macromer backbone region is formed of a degradable backbone region having water-soluble regions as branches or grafts attached to the degradable backbone. Two or more polymerizable regions are attached to the water soluble branches or grafts.

In another variation, the macromer backbone may have multiple arms; e.g., it may be star-shaped or comb-shaped. The backbone may include a water-soluble region, a biodegradable region, or a water-soluble, biodegradable region. The polymerizable regions are attached to this backbone. Again, the polymerizable regions must be separated at some point by a degradable region.

Throughout the specification, the following abbreviations are sometimes used to describe the specific macromers of the invention. In three particular examples, a macromer having a water soluble region consisting of PEG with a molecular weight of 4,000 daltons, with 5 lactate groups on either side of this region, capped on either side with acrylate groups, is referred to as "4kL5." Similarly, a macromer having a water soluble region consisting of PEG with a molecular weight of 3,400 daltons, with 6 caprolactone groups on either side of this region, capped on either side with acrylate groups, is referred to as "3.4kC6." Likewise, a macromer having a water soluble region consisting of PEG having a molecular weight of 5,400 daltons and 3 arms, each arm containing 3 lactate groups, extending from this region, capped on either side with acrylate groups, is referred to as "4.2kL3-A3."

Water-Soluble Region

In preferred embodiments, the central core is a water soluble region. This water soluble region of the macromer may include poly(ethylene glycol), poly(ethylene oxide), poly(vinyl alcohol), poly(vinylpyrrolidone), poly(ethyloxazoline), poly(ethylene oxide)-co-poly(propylene oxide) block copolymers, polysaccharides, carbohydrates, or proteins, or combinations thereof.

The macromer preferably comprises a water soluble core region comprising PEG, as PEG has high hydrophilicity and water solubility, as well as good biocompatibility. The PEG region preferably has a molecular weight of about 400 to about 40,000 daltons, and more preferably has a molecular weight of about 1,000 to about 30,000 daltons, about 1,000 to about 20,000 daltons, or about 2,000 to about 10,000 daltons.

Degradable Region

The degradable region of the macromer may contain, for example, poly(α-hydroxy acids), poly(lactones), poly(amino acids), poly(anhydrides), poly(orthoesters), poly(orthocarbonates) or poly(phosphoesters), or blends or copolymers of these polymers.

Exemplary poly(α-hydroxy acids) include poly(glycolic acid), poly(DL-lactic acid), and poly(L-lactic acid). Exemplary poly(lactones) include poly(ε-caprolactone), poly(δ-valerolactone), poly(γ-butyrolactone), poly(1,5-dioxepan-2-one), and poly(trimethylene carbonate).

The degradable region may comprise a blend of at least two different polymers. Examples of copolymers include a copolymer of caprolactone and glycolic acid; and a copolymer of caprolactone and lactic acid.

Polymerizable Region

The polymerizable regions of the macromer preferably contain carbon-carbon double bonds capable of polymerizing the macromers. The choice of an appropriate polymerizable group permits rapid polymerization and gelation. Polymerizable regions containing acrylates are preferred because they can be polymerized using several initiating systems, as discussed below. Examples of acrylates include acrylate, methacrylate, and methyl methacrylate.

Polymerization of Macromers

If desired, the macromers of the present invention may be polymerized using polymerization initiators under the influence of long wavelength ultraviolet light, visible light, thermal energy, or a redox system. The polymerization can be conducted at room temperature or at lower temperatures, for example, temperatures less than 20° C. During polymerization, substances such as proteins are physically incorporated into the resulting polymer network of the hydrogel.

Polymerization of the macromers may be initiated in situ by light having a wavelength of 320 nm or longer. When the polymerizable region contains acrylate groups, the initiator may be any of a number of suitable dyes, such as xanthine dyes, acridine dyes, thiazine dyes, phenazine dyes, camphorquinone dyes, acetophenone dyes, or eosin dyes with triethanolamine, 2,2-dimethyl-2-phenyl acetophenone, and 2-methoxy-2-phenyl acetophenone.

The polymerization may also take place in the absence of light. For example, the polymerization can be initiated with a redox system, using techniques known to those of skill in the art. In some cases it is advantageous to polymerize macromers using the redox system of the invention, as radical initiator production occurs at reasonable rates over a wide range of temperatures.

Initiators that can be used in the redox system include, without limitation, peroxides such as acetyl, benzoyl, cumyl and t-butyl; hydroperoxides such as t-butyl and cumyl, peresters such as t-butyl perbenzoate; acyl alkylsulfonyl peroxides, dialkyl peroxydicarbonates, diperoxyketals, ketone peroxide, azo compounds such as 2,2′-azo(bis)isobutyronitrile (AIBN), disulfides, and tetrazenes.

Shaping of Articles

The articles of the present invention may be formed in any shape desired. For example, the articles may be shaped to fit into a specific body cavity. They may also be formed into thin, flat disks or particles, such as microspheres. Alternatively, the articles may be shaped, then processed into the desired shape before use, or ground into fine particles. The desired shape of the article will depend on the specific application.

Macromer particles may be prepared using techniques known in the art, including single and double emulsion solvent evaporation, spray drying, and solvent extraction. As used herein, the term "particles" includes, but is not limited to, microspheres. In a microsphere, a BAS is dispersed throughout the particle. The particles may have a smooth or irregular surface, and may be solid or porous. Methods for making microspheres are described in the literature, for example, in U.S. Pat. No. 4,272,398, Mathiowitz and Langer (J. Controlled Release 5:13-22 (1987)); Mathiowitz et al. (Reactive Polymers 6:275-283 (1987)); Mathiowitz et al. (J. Appl. Polymer Sci. 35:755-774 (1988)); Mathiowitz et al. (Scanning Microscopy 4:329-340 (1990)); Mathiowitz et al. (J. Appl. Polymer Sci., 45:125-134 (1992)); and Benita et al. (J. Pharm. Sci. 73:1721-1724 (1984)), hereby incorporated by reference. In one preferred embodiment of the present invention, the microspheres are formed into hydrogel droplets.

In solvent evaporation, described, for example, in Mathiowitz, et al., (1990), Benita et al. (1984), and U.S. Pat. No. 4,272,398, a polymer is dissolved in a volatile organic solvent, such as methylene chloride. An agent to be incorporated, either in soluble form or dispersed as fine particles, is optionally added to the polymer solution, and the mixture is suspended in an aqueous phase that contains a surface active agent such as poly(vinyl alcohol). The resulting emulsion is stirred until most of the organic solvent evaporates, leaving solid microspheres, which may be washed with water and dried overnight in a lyophilizer.

In solvent removal, as described, for example, by Park et al. (J. Controlled Release 55:181-191 (1998)), a therapeutic or diagnostic agent is dispersed or dissolved in a solution of a selected polymer in a volatile organic solvent such as methylene chloride. The mixture can then be suspended in oil, such as silicon oil, by stirring, to form an emulsion. As the solvent diffuses into the oil phase, the emulsion droplets harden into solid polymer microspheres.

Processes for preparing ultrafine particles of biological molecules by atomizing liquid solutions of the macromolecules, drying the droplets formed in the atomization step, and collecting the particles are described in PCT WO 97/41833, hereby incorporated by reference.

Spray drying is implemented by passing a homogenous mixture of a BAS, such as a therapeutic agent, and the polymerizable macromer used to form a hydrogel through a nozzle, spinning disk, or equivalent device to atomize the mixture to form fine droplets. The substance and the polymerizable macromer may be provided in a solution or suspension, such as an aqueous solution. The fine droplets are exposed to light to cause polymerization of the macromer and formation of the hydrogel droplets incorporating the substance. Hydrogels may be formed according to the methods described in U.S. Pat. No. 5,410,016, hereby incorporated by reference, or other techniques known in the art of polymer chemistry.

In another embodiment, hydrogel particles are prepared by a water-in-oil emulsion process, wherein the polymerizable macromers and the substance to be incorporated are suspended in a water-in-oil emulsion and exposed to light to polymerize the macromers to form hydrogel particles incorporating the substance, such as a BAS. Typically, polymerization may be conducted at room temperature.

The microspheres prepared using the techniques described above are freeze dried, so they have a long shelf life (without biodegradation) and the BAS remains biologically active. Prior to use for injectable formulations, the microspheres are reconstituted in a suitable solution, such as saline or other liquids. For pulmonary delivery, either freeze dried or reconstituted particles may be used.

Properties of the Macromers

The articles of the present invention are biodegradable. Biodegradation occurs at the linkages within the extension oligomers and results in fragments which are non-toxic and easily removed from the body and/or are normal, safe chemical intermediates in the body. These materials are particularly useful for the delivery of hydrophilic materials, since the water soluble regions of the polymer allow water to access the materials trapped within the polymer.

Use of the Macromers

Macromers can be shaped into articles, for example, microspheres, and these articles are capable of degrading under in vivo conditions at rates which permit the controlled release of incorporated substances. Release of such a substance may occur by diffusion of the substance from the polymer prior to degradation and/or by diffusion of the material from the polymer as it degrades. Degradation of the polymer facilitates eventual controlled release of free macromolecules in vivo by gradual hydrolysis of the terminal ester linkages. The burst effects that are sometimes associated with other release systems are thus avoided in a range of formulations.

The rate of release of a BAS depends on many factors, for example, the composition of the water soluble region, the degree of polymerization of the macromer. The rate of release of a BAS also depends on the rate of degradation of the degradable region of the macromer. For example, glycolic esters lead to very rapid degradation, lactic esters to somewhat slower degradation, and caprolactic esters to very slow degradation. When the degradable region consists of polyglycolic acid, the release period is less than one week. When the degradable region consists of poly(lactic acid), the release period is about one week. When the degradable region consists of a copolymer of caprolactone and lactic acid or a copolymer of trimethylene carbonate and lactic acid, the release period is two to four weeks. When the degradable region consists of poly(trimethylene carbonate) or a copolymer of caprolactone and trimethylene carbonate, the release period is about three to eight weeks. When the degradable region consists of poly(trimethylene carbonate) or poly(caprolactone), the release period is longer than about five weeks.

The precise rate of release of a BAS from an article can be further modified by altering the ratio of hydrophilic and hydrophobic components of the article. For example, a very soluble macromer will yield, after polymerization, a hydrophilic gel; hydrophilic hydrogels have been shown to degrade more rapidly than hydrophobic ones. A blend of a hydrophilic macromer (e.g., 4kL5) with a hydrophobic water insoluble macromer (3.4kC6) is used to form a polymerized hydrogel. This hydrogel will have a release rate that is in between the release rate of a hydrogel containing only lactic acid and a hydrogel containing only caprolactone. A macromer in which the degradable region is a copolymer of caprolactone and lactic acid will also have a release rate which is in between the release rate of a hydrogel containing only lactic acid and a hydrogel containing only caprolactone as the primary degradable group. Similarly, hydrophilicity of the active substance also affect the release rate of the BAS, with hydrophilic active substances generally released faster than hydrophobic substances.

The rate of release of a given BAS from a therapeutic article depends on the quantity of the loaded substance, as a percent of the final product formulation. For example, it is generally thought in the polymer field that while a large amount of BAS loading results in a longer period of therapeutic dose delivery, it also results in a large burst effect. Therefore, an article which is loaded with a high amount of a BAS, and which also exhibits a low burst effect would be an optimal article. The articles or the present invention exhibit these characteristics.

Other factors which affect the release rate of a BAS from an article are the aggregation and the solubility of the BAS. In order for the articles of the present invention to have release profiles which are optimal for delivering a BAS, the percent of the BAS which is aggregated should be low. The articles of the present invention contain BAS which are preferably less than 15% aggregated. In preferred embodiments, the articles have this characterization of low aggregation even when they and contain at least 2.5% BAS by dry weight, more preferably at least 5%, and most preferably 20 or 40% by dry weight.

As stated above, another factor which affects the rate of release of a BAS from an article is the solubility of the BAS in the article. In the field of polymer chemistry, it has generally been thought that water-soluble substances, such as a BAS, will yield homogenous systems when incorporated into the macromers of the invention. It has also been thought that substances that do not solubilize in water within the time it takes to form the macromers of the invention will yield heterogenous systems. While the amount of burst in the heterogenous systems can be minimized by using a particulate suspension with small particles, it is generally thought that substances should be in a water soluble format for optimal delivery in a polymer delivery system. The articles of the present invention contain a BAS in an insoluble format, and these articles exhibit a low burst effect, an unexpected result.

Yet another factor that affects the release rate of a BAS from an article is the particle size of the BAS. For example, the articles of the present invention feature a BAS which has been ground and sieved to isolate fine particles which are smaller than approximately 75 microns in any dimension. These particles were used to generate microspheres and the release of the BAS from the microspheres was measured. This release rate was compared to the release rate of the same BAS from the same microspheres, with the exception that the BAS was not fine-ground. The results of these studies indicated that a BAS which is fine-ground results in release rates which are slower and have a low burst effect. By adjusting the factors discussed above, degradation and controlled release may be varied over very wide ranges. For example, release may be designed to occur over hours, days, or months.

The methods of the invention can produce particles that behave as homogenous drug delivery systems. Because of the homogenous nature of the articles of the invention, there is no initial burst of released substance. In addition, the uniform consistency makes it possible to incorporate relatively high amounts of protein, while still minimizing the burst effect.

The present invention also features insoluble macromers. These macromers contain at least one water-soluble region, at least one degradable (e.g., hydrolyzable) region, and at least one polymerizable region. The degradable region contains polymers of glycolic acid, lactic acid, caprolactone, trimethylene carbonate, or blends or copolymers thereof. The degradable region must be water insoluble. For example, a macromer having a degradable region containing 15-20 lactide units can be prepared; this macromer will provide a relatively fast release rate. A macromer with a degradable region containing 6 caprolactone units will provide a relatively slow release rate. A macromer with a degradable region containing a copolymer of 6 caprolactone units, 4 lactide units, and 4 glycolide units will provide a fast release rate, and a macromer with a degradable region containing a copolymer of 3 lactide units and 7 trimethylene carbonate units will provide an intermediate release rate.

The water soluble region of these macromers is preferably PEG. The water soluble region can have multiple arms; for example, it may be star-shaped or comb-shaped, as described, for example in U.S. Pat. No. 5,410,016, incorporated herein by reference. The water soluble region preferably has 3, 4, 6, or 8 arms and a molecular weight of 500 to 20,000, preferably, 1,000 to 10,000 daltons.

Methods for Increasing Protein Precipitation

The articles of the present invention, can be made to contain a BAS in an insoluble format, by combining the BAS with a molecule, or mixture or molecules which preferentially excludes proteins, and a macromer, forming a mixture of these reagents, and polymerizing the mixture. A molecule or mixture of molecules which preferentially exclude proteins can be used in the formation of the article to increase protein precipitation. Examples of molecules which preferentially exclude proteins include, but are not limited to, macromers, poly(ethylene glycol), hyaluronic acid, and poly(vinylpyrrolidone). A reagent which carries a positive or negative ion charge may be used in the formation of the articles of the invention in order to increase the precipitation of the BAS in the mixture which is then polymerized to form the article. The optimal reagent to be used depends on the charge of the protein, which is affected by the pH of the mixture. Examples of mixtures of molecules which preferentially exclude proteins include, but are not limited to, a mixture of molecules comprising a positively charged ion-carrying reagent, for example, triethanolamine or Tris (for example, when the pH is such that the protein is negatively charged); or a mixture of molecules comprising a negatively charged ion-carrying reagent, such as sodium dodecyl sulfate (for example, when the pH is such that the protein is positively charged). A mixture comprising a surfactant, for example, Tween 20, Tween 80, or poloxamer F68, may also be used to increase the precipitation of the protein.

High Load and Low Burst Characteristics

A therapeutic agent, for example, a BAS may be readily incorporated in high yield into the articles described herein. For example, articles may be prepared containing at least 5% active substance by dry weight. Preferably, the articles contain at least 10, 25, or 40% by dry weight.

As discussed above, the BAS of the present invention is in an insoluble format when combined with a macromer and formed into an article. The combination of high load and the insoluble format of the active substance in the article provides the article with a slow release profile, with little initial burst. These results are surprising given the view in the field of polymers that an article containing an insoluble active substance will have large initial burst of the active substance.

The BAS contained in the articles of the present invention is insoluble. The formulation of articles containing an insoluble BAS may be achieved, for example, by mixing the BAS with PEG, and then combining these reagents with the desired macromer.

The amount of BAS loaded into a microsphere may be measured by combining it with a macromer and shaping into articles. The articles may then be placed into an appropriate solvent, for example phosphate buffered release media (0.01% NaN₃, 0.05 M PBS, pH 7.4) and assayed for the amount of BAS present by means available in the art, such as spectrophotometry.

Biologically Active Substances

A BAS that can be incorporated into the articles of the invention include therapeutic, diagnostic, and prophylactic agents. They can be naturally occurring compounds, synthetic organic compounds, or inorganic compounds. Substances that can be incorporated into the articles of the invention include proteins, polypeptides, carbohydrates, inorganic materials, antibiotics, antineoplastic agents, local anesthetics, antiangiogenic agents, vasoactive agents, anticoagulants, immunomodulators, cytotoxic agents, antiviral agents, antibodies, neurotransmitters, psychoactive drugs, oligonucleotides, lipids, cells, tissues, tissue or cell aggregates, and combinations thereof.

Exemplary therapeutic agents include growth hormone, for example human growth hormone, calcitonin, granulocyte macrophage colony stimulating factor (GMCSF), ciliary neurotrophic factor, parathyroid hormone, and the cystic fibrosis transmembrane regulator gene. Other specific therapeutic agents include parathyroid hormone-related polypeptide, somatostatin, testosterone, progesterone, estradiol, nicotine, fentanyl, norethisterone, clonidine, scopolomine, salicylate, salmeterol, formeterol, albeterol, and valium.

Drugs for the treatment of pneumonia may be used, including pentamidine isethionate. Drugs for the treatment of pulmonary conditions, such as asthma, may be used, including albuterol sulfate, β-agonists, metaproterenol sulfate, beclomethasone dipropionate, triamcinolone acetamide, budesonide acetonide, ipratropium bromide, flunisolide, cromolyn sodium, ergotamine tartrate, and protein or polypeptide drugs such as TNF antagonists or interleukin antagonists.

Other therapeutic agents include cancer chemotherapeutic agents, such as cytokines, chemokines, lymphokines, and substantially purified nucleic acids, and vaccines, such as attenuated influenza virus. Substantially purified nucleic acids that can be incorporated include genomic nucleic acid sequences, cDNAs encoding proteins, expression vectors, antisense molecules that bind to complementary nucleic acid sequences to inhibit transcription or translation, and ribozymes. For example, genes for the treatment of diseases such as cystic fibrosis can be administered. Polysaccharides, such as heparin, can also be administered.

Other therapeutic agents include tissue plasminogen activator (t-PA), superoxide dismutase, catalase luteinizing hormone releasing hormone (LHRH) antagonists, IL-11 platelet factor, IL-4 receptor, enbrel, IL-1 receptor antagonists, TNF receptor fusion proteins, megakaryocyte growth and development factor (MGDF), stemgen, anti-HER-2 and anti-VEGF humanized monoclonal antibody, anti-Tac antibody, GLP-1 amylin, and GLP-1 amylin analogues.

Additional therapeutic agents include atrial natriuretic factor, atrial natriuretic peptide, beta-human chorionic gonadotropin, basic fibroblast growth factor, bovine growth hormone, bone morphogenetic protein, B cell stimulating factor-1, B cell stimulating factor-2, bovine somatotropin, carcinobreaking factor, cartilage induction factor, corticotropin releasing factor, colony stimulating factor, differentiating factor-1, endothelial cell growth factor, erythroid differentiation factor, elongation factor 1-alpha, epidermal growth factor, erythropoietin, thrombopoietin, thymopoietin, fibroblast growth factor, follicle stimulating hormone, granulocyte colony stimulating factor, glial fibrillary acidic protein, growth hormone releasing factor, human alpha-1 antitrypsin, human atrial natriuretic factor, human chorionic gonadotropin, human leukemia inhibitory factor, hemopoietin-1, hepatocyte growth factor, human transforming growth factor, human thyroid-stimulating hormone, interferon, immunoglobulin A, immunoglobulin D, immunoglobulin E, insulin-like growth factor-1, insulin-like growth factor-II, immunoglobulin G, immunoglobulin M, interleukin-1, interleukin-2, interleukin-3, interleukin-4, interleukin-5, interleukin-6, kidney plasminogen activator, lectin cell adhesion molecule, luteinizing hormone, leukemia inhibitor factor, monoclonal antibody, macrophage activating factor, macrophage cytotoxic factor, macrophage colony stimulating factor, megakaryocyte colony stimulating factor, tumor necrosis factor, macrophage inhibitory factor, Mullerian inhibiting substance, megakaryocyte stimulating factor, melanocyte stimulating factor, neutrophil chemotactic factor, nerve growth factor, novel plasminogen activator, nonsteroidal anti-inflammatory drug, osteogenic factor extract, antitumor lymphokine, prostate-specific antigen, anti-platelet activating factor, plasminogen activator inhibitor, platelet-derived growth factor, platelet-derived wound healing formula, plasmatic human interleukin inducing protein, tumor angiogenesis factor, tissue control factor, T cell growth factor, T cell modulatory peptide, transforming growth factor, tumor growth inhibitor, tumor inhibiting factor, tissue inhibitor of metalloproteinases, tumor necrosis factor, tissue plasminogen activator, thyroid stimulating hormone, urokinase-plasminogen activator, vascular endothelial growth factor, and vasoactive intestinal peptide.

A preferred BAS is a substantially purified polypeptide or protein. Proteins are generally defined as consisting of 100 amino acid residues or more; polypeptides are less than 100 amino acid residues. Unless otherwise stated, the term protein, as used herein, refers to both proteins and polypeptides. The proteins may be produced, for example, by isolation from natural sources or recombinantly. Examples include insulin and other hormones, including growth hormones, such as human growth hormone and bovine growth hormone. Other exemplary proteins include Factor VIII, Factor IX, Factor VIIa, and anti-inflammatory agents, such as interleukins, including interleukin-4. Other exemplary proteins include enzymes, such as DNase and proteases. Other proteins include cytokines, interferons, including interferon alpha and interferon beta, poetins, angiogenic factors, differentiation factors, colony-stimulating factors, growth factors, ceredase, gibberellins, auxins, and vitamins, and fragments thereof. Exemplary growth factors include vascular endothelial growth factor (VEGF), endothelial cell growth factor (ECGF), basic fibroblast growth factor (bFGF), and platelet derived growth factor (PDGF).

Proteins are stable in the hydrogels of the present invention. For example, many of the proteins are protected from dimerization or aggregation, as discussed below in the Examples. The enzymatic degradation of proteins or polypeptides can be further minimized by co-incorporating peptidase-inhibitors.

Treatment of an Animal Using Slow Release Protein Polymers

The polymer articles of the present invention may be used to treat an animal, for example, a mouse, rat, or human, by delivering a BAS to the animal. The articles may contain such a BAS as any of those described above. Various routes of administration may be used to deliver the articles of the present invention, as described below.

The results of the treatment of an animal with therapeutic articles containing a BAS, as described herein, will vary according to the BAS being delivered. For example, if hGH is delivered through the therapeutic articles of the present invention, one would expect to observe an increase in growth as a result of such a treatment. If erythropoietin is delivered through the therapeutic articles, one would expect to observe an increase in reticulocytes in the animal as a result of the treatment. If insulin is delivered through the therapeutic articles, then the treatment should result in a decrease in blood glucose levels.

The articles of the present invention provide optimal delivery of a BAS, because they release the BAS in a controlled manner with a low burst effect. The result of such a delivery rate is that the drug is delivered steadily over a desired period of time. A slower and steadier rate of delivery may in turn result in a reduction in the frequency with which the BAS must be administered to the animal. In addition, a low burst effect may be highly desirable in some circumstances where the delivery of too much BAS to a site is deleterious to the animal.

Routes of Administration of the Articles

Inhalation

The use of the hydrogel particles of the invention can enhance the delivery of drugs to the lung. Administration to the lung provides for the delivery of drugs that can be transported across the lung tissue barriers and into circulation, as described WO 99/03454.

A problem with the delivery of active substances to the lung is that pulmonary macrophages can take up the materials, thus preventing the material from entering into systemic and local circulation. Uptake occurs when proteins adsorbed to the particles' surfaces bind with receptors on the surfaces of the macrophages. To prevent uptake, the invention provides nonionic hydrogels, e.g., formed with polymers based on polyethylene glycol. These hydrogels adsorb low levels of proteins and thus bind poorly to cell surfaces. Anionic hydrogels, e.g., formed with polyacrylic acid, also adsorb relatively low levels of proteins and thus bind poorly to cell surfaces.

In a further embodiment, biocompatible microcapsules may be formed and the surface provided with water soluble non-ionic polymers such as polyethylene oxide (PEO), to create resistance to cell adhesion, as described in U.S. Pat. No. 5,380,536, hereby incorporated by reference.

The size and density of the particles can also be selected to maximize the quantity of BAS that is delivered to the lung. For example, the macrophages will not take up large particles as efficiently as they will take up small particles. However, large particles are not delivered to the deep lung as well as small particles are. To overcome these conflicting factors, the invention provides small particles that can swell as they hydrate. The particles are administered to the deep lung as small (i.e., 1-5 microns), dry, or slightly wet, particles; upon hydration, they swell, and therefore become resistant to uptake by the pulmonary macrophages. The swelling can occur when the particles are hydrated from the dry state and when they are hydrated from one state of hydration to another by a change in temperature, pH, salt concentration, or the presence of other solvents, for example, depending upon the chemical and physical nature of the hydrogel polymer.

As used herein, the term "dry" means that the particles of the powder have a moisture content such that the powder is readily dispersible in an inhalation device to form an aerosol. Preferably, the moisture content of the particles is below 10% by weight water, more preferably below about 5%, or optionally below about 2%, or lower.

The density of the particles is expressed in terms of tap density. Tap density is a standard measure of the envelope mass density. The envelope mass density of an isotropic particle is defined as the mass of the particle divided by the minimum sphere envelope volume within which it can be enclosed. The density of particles can be measured using a GeoPyc (Micrometers Instrument Corp., Norcross, Ga.) or a AutoTap (Quantachrome Corp., Boyton Beach, Fla.).

For example, the density of 3.4kL5 particles was determined as follows. 3.4kL5 (1.0025 g), 200 mM TEOA in PBS; pH 7 (1.0260 g), and 1000 ppm Eosin (0.1028 g) were combined. 200 mg of this solution was mixed with talc (0.1015 g). The resulting suspension was placed in a 100 μl glass pipet and polymerized by light for 15 seconds (ILC Technology, Inc. Xenon Light Source with Fiber Optics). The rod was pushed out, placed on aluminum foil, and further polymerized for 3.5 minutes. The hardened rod was lyophilized (vacuum 15E-3 mbar, trap temp. -50° C.) for 18 hours. The dry rod (water content<10%) was cut into small pieces, placed in heptane, and minced using a homogenizer (Silverson L4RT-A) at 5,000 rpm to small particles. The wet particles were air-dried, followed by nitrogen gas flow. The particles sizes ranged from 1 micron to 0.5 mm.

1.645 g of these particles was placed in a 10 mL graduated cylinder. The graduated cylinder was mounted on top of an Autotap densimeter (Quantachrome). The sample was tapped 100 times and the particles' volume was read. The process was repeated until no change in volume was observed. The final volume was 2.8 ml. The tap density of the particles was 1.6435 g/2.8 ml=0.5870 g/ml.

In addition to particles, the polymer may be provided in other shapes suitable for delivery to the deep lung. For example, PEG emulsion microspheres are subjected to high pressure and a vacuum onto a flat plate to form very light very thin layers, for example, having a snow flake consistency, that react differently to fluidic wind forces. The resulting thin flakes can be, e.g., 0.01 micron, 1 micron, or 10 microns thick.

The particles can be administered to the respiratory system alone, or in any appropriate pharmaceutically acceptable excipient, such as a liquid, for example, saline, or a powder. Aerosol dosages, formulations and delivery systems may be selected for a particular therapeutic application, as described, for example, in Gonda ("Aerosols for delivery of therapeutic and diagnostic agents to the respiratory tract," in Critical Reviews in Therapeutic Drug Carrier Systems, 6:273-313, 1990); and in Moren ("Aerosol dosage forms and formulations," in: Aerosols in Medicine. Principles, Diagnosis and Therapy, Moren, et al., Eds., Elsevier, Amsterdam, 1985).

Pulmonary drug delivery may be achieved using devices such as liquid nebulizers, aerosol-based metered dose inhalers, and dry powder dispersion devices. For the use of dry powder dispersion devices, the polymer particle incorporating the therapeutic agent is formulated as a dry powder, for example, by lyophilization or spray-drying. Methods for preparing spray-dried, pharmaceutical-based dry powders including a pharmaceutically acceptable amount of a therapeutic agent and a carrier are described in PCT WO 96/32149, hereby incorporated by reference.

Examples of a BAS that can be administered to the lung include, without limitation, insulin, antitrypsin, calcitonin, alpha interferon, beta interferon, GLP-1, and DNAse.

Nasal Delivery

The articles of the present invention can also be used to administer compounds nasally. For example, a vaccine containing freeze dried or reconstituted microspheres can be administered nasally.

Intramuscular and Subcutaneous Administration

The articles of the present invention can be used to administer microspheres that degrade over several days to 3 months, by intramuscular injection or by subcutaneous injection.

For example, growth hormone can be administered subcutaneously; the hormone leaves the microspheres at the site of injection as they degrade. Growth hormone enters the systemic circulation, where, in turn, it exerts its effects directly, and indirectly through induction of somatomedin production in the liver and in other tissues. For this application, particle sizes of up to 0.5 mm can be used.

In other embodiments, the active agent is a vaccine, such as tetanus vaccine, other proteins or polypeptides, or more complex immunogens. The vaccine is released over time, from one week to many weeks, resulting in an improved immune response to the vaccine, compared to a bolus injection followed by one or more booster shots with the same total dose of immunogen. Mixtures of different types of microspheres can result in initial and booster shot-type immunization as well.

Intravenous Administration

Articles that contain a BAS useful in treating clotting disorders, such as Factor VIII or Factor IX for hemophilia, can be administered by intravenous injection. The BAS is released over days to weeks. A therapeutic level of the BAS is maintained that results in a better clinical outcome. In addition, potentially lower total doses of a BAS can be administered, with a corresponding economic benefit. These approaches help promote patient compliance.

In the case of intravenous injection, it is important to formulate the microspheres in acceptable agents so the microspheres do not aggregate and clog blood vessels. The microspheres must be appropriately sized, so that they don't lodge in capillaries. For this application, particle sizes of 0.2-0.5 microns are preferred.

In a number of inflammatory conditions, as part of the inflammatory process that is mediated by selectin and ICAM expression/binding with neutrophil intravisation, blood vessels become leaky at the site of inflammation. Hydrogel microspheres may be administered; these microspheres will leak out of blood vessels at the site of inflammation, and then release their BAS payload locally over a period of time. Disease conditions where this approach may be useful could include, but are not limited to, inflammatory bowel diseases, asthma, rheumatoid arthritis, osteoarthritis, emphysema, and cystic fibrosis (with DNase as the enzymatic drug).

Hydrogel microspheres that contain cytokines, lymphokines, or other compounds to treat cancer can be administered by intravenous injection. Blood vessels within large solid tumors are generally leaky, and the blood flow within them is often slow. Thus, microspheres could lodge within solid tumors and release their anticancer BAS locally, either killing tumor cells directly or by activating the immune system locally. This approach could be used, for example, with compounds such as interleukin 2, where the systemic and local toxicity has been dose limiting and where the resulting side effects are significant.

The microspheres of the present invention may be cleared relatively slowly from the circulation. Alternatively, the microspheres can be targeted to exit the circulatory system through leaky blood vessels or through more active targeting mechanisms, e.g., receptor mediated targeting mechanisms.

Oral Administration

In some portions of the gastrointestinal tract, there is relatively good transport of proteins across the intestinal mucosa into the systemic and local circulation. The compositions of the invention, for example, freeze dried microspheres containing protein (with very small particle sizes), can therefore be administered orally in an appropriate enteric formulation that protects the drug-containing microspheres from enzymatic attack and the low pH found in the upper GI tract. Such an enteric formulation could also be designed using several available technologies to gradually expel BAS-containing microspheres as the enteric capsule traverses the gastrointestinal tract. This is described in more detail in WO 99/03454 and in Mathiowitz et al. (Nature 386: 410-414 (1997)). It is anticipated that this approach will have a number of advantages over other approaches for delivering proteins and other molecules, even small molecules, orally. First, PEG and proteins are compatible, so the major manufacturing and stability problems found with other drug delivery approaches can be avoided. Secondly, dried hydrogels are very adhesive to wet tissue. The microparticles will bind well to the GI tract and will be transported into the system via the gastrointestinal circulation or release their contents on the intestinal mucosa; in turn, the drug will enter the systemic and gastrointestinal circulation. Chemical enhancers, or formulations containing compositions that utilize specific and non-specific biological transport mechanisms to facilitate transport across the GI tract into the systemic circulation, can be included as well.

Targeting

Targeting ligands can be attached to the particles via reactive functional groups on the particles. Targeting ligands permit binding interactions of the particle with specific receptor sites, such as those within the lungs or those on endothelial cells specific to different regions in the body's microvasculature. A targeting ligand is selected which specifically or non-specifically binds to particular targets. Exemplary targeting ligands include antibodies and fragments thereof including antibody variable regions, lectins, hormones, or other organic molecules capable of specific binding to receptors on the surfaces of the target cells. Other ligands are described in Science (279:323-324 (1998)), hereby incorporated by reference.

Microspheres can be made with both a BAS and a targeting molecule. Double microspheres can also be made, in which the inner sphere contains drug and the outer PEG shell contains the targeting molecule or reagent.

Excipients and Carriers

The particles incorporating a therapeutic agent or diagnostic agent may be provided in combination with one or more pharmaceutically acceptable excipients available in the art, as described, for example, in PCT WO 95/31479, hereby incorporated by reference. Excipients may be selected that can, in some applications, enhance stability, dispersability, consistency, and bulking to ensure uniform pulmonary delivery. The excipient may be, e.g., human serum albumin (HSA), bulking agents such as carbohydrates, amino acids, polypeptides, pH adjusters or buffers, and salts. Additional excipients include zinc, ascorbic acid, mannitol, sucrose, trehalose, cyclodextrans, polyethylene glycol, and other commonly used pharmaceutical excipients, including those described in The United States Pharmacopeia, published by the United States Pharmacopeia Convention, Inc., 1995 (see, e.g., pp. 2205-2207). Exemplary carbohydrates include monosaccharides, such as galactose, and disaccharides such as lactose. Excipients that stabilize proteins are especially useful.

In some cases, the excipients are used as carriers; i.e., they are used to modulate the release rate of the active substances. For example, mannitol can be used to accelerate or delay release.
 

Claim 1 of 16 Claims

1. A biocompatible therapeutic article comprising, a macromer having polymerized end groups, precipitated human growth hormone, and a molecule or mixture of molecules which preferentially excludes proteins, wherein said molecule or mixture of molecules is present in an amount sufficient to reduce the solubility of said human growth hormone in said article to less than 10 mg/ml.

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