CZ2000218A3 - Biologically active compound intended for therapy, preparation for administering the biologically active compound, preparation process, and insoluble macromer and hydrogel preparation - Google Patents

Abstract

The present invention relates to a biologically active substance for use in treatment, a means for delivering a biologically active agent, a process for the production of insoluble macromer and hydrogel a composition made of biodegradable macromer. A chemical composition of the macromer is possible to influence and control the release rate biologically active compounds in the body.

Description

Biologically active agent for use in therapy, agent for delivering biologically active agent, method of manufacture, insoluble macromer and hydrogel agent

Technical field

The invention relates to methods of administering biologically active compounds and biodegradable compositions for administering such compounds.

BACKGROUND OF THE INVENTION

Rapid advances in the field of genetic engineering and biotechnology have led to the development of an increasing number of proteins and peptides that are suitable as pharmaceutical agents. Thus, the development of methods of administering these new pharmaceutical agents is becoming increasingly important. In particular, topical or systemic administration of biologically active substances such as proteins is of particular interest.

Protein delivery can be complicated because proteins will degrade using many carriers that have traditionally been used for the administration of small molecules. In many cases, it is difficult to convert active forms of proteins into biodegradable polymers. Synthetic materials such as biodegradable hydrogels can be used for protein delivery. However, in many methods, protein delivery to systemic and local circulation is relatively rapid and is primarily determined by the rate of dissolution of protein particles. These methods may be of limited use since drug release may occur in a steep manner instead of a steady, controlled rate.

SUMMARY OF THE INVENTION

First, the invention features a method of delivering a biologically active agent, comprising the steps of: (a) combining the active agent with a macromer; (b) forming a mixture of the combinations formed in step (a); (c) polymerizing the mixture to form preparations; and administering the compositions or a portion thereof to the mammal, wherein step (c) proceeds in the absence of a polymerizable monovinyl monomer.

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Second, the invention features a method of delivering a biologically active agent, comprising the steps of: (a) combining the active agent with a macromer; (b) forming a mixture of the combinations formed in step (a); (c) polymerizing the mixture to form preparations; and administering the compositions or a portion thereof to a mammal, wherein step (c) proceeds in the absence of a water-soluble polymerizable monovinyl monomer.

Third, the invention features a method of delivering a biologically active agent, comprising the steps of: (a) combining the active agent with a macromer; (b) forming a mixture of the combinations formed in step (a); (c) polymerizing the mixture to form preparations; and administering the compositions or a portion thereof to the mammal, wherein step (c) proceeds in the absence of vinylpyrrolidone monomer. The invention also features compositions produced by these methods.

Fourthly, the invention features a method of delivering a biologically active agent, comprising the steps of: (a) combining the active agent with a macromer; (b) forming a mixture of the combinations formed in step (a); (c) polymerizing the mixture to form preparations; and administering the compositions or a portion thereof to a mammal, wherein the compositions release at least 80% of the active agent over a period of 2.5 times longer than t ₅₀ .

Fifthly, the invention features a method of delivering a biologically active agent, comprising the steps of: (a) combining the active agent with a macromer; (b) forming a mixture of the combinations formed in step (a); (c) polymerizing the mixture to form preparations; and administering the compositions or portions thereof to a mammal, wherein the compositions release a therapeutic dose of the active agent over a period of at least 2.5 times longer than the t 50.

Sixth, the invention provides a composition for delivering a biologically active agent, a composition comprising particles comprising a hydrogel and a biologically active agent, wherein the release kinetics of the particles are independent of particle size, the particles having a mean diameter of 50 nm to 1 mm.

Seventh, the invention features a method of making a controlled release formulation of a biologically active agent, comprising the steps of: (a) combining the active agent with a biodegradable, polymerizable macromer, wherein the macromer comprises at least one water soluble region, at least one degradable region that is hydrolyzable; in vivo conditions and a polymerizing end group having the ability to form additional covalent bonds resulting in polymerization of the macromer, wherein the polymerizing end groups are separated by at least one degradable region, in the presence of an initiator; (b) Polymerization 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 A macromer in the absence of light to form a hydrogel and incorporate the active ingredient into the hydrogel; and (c) transforming the hydrogel into formulations capable of controlled release of the active agent. The initiator may be a free radical initiator or an ionic initiator.

Eighth, the invention features a method of preparing a polymerized hydrogel, the method comprising the steps of: (a) combining a hydrophobic, water-insoluble macromer, initiator, and water; (b) allowing the macromer to swell; (c) mixing the macromer to form a homogeneous mixture; and (d) polymerizing the macromer and forming a hydrogel. Preferably, the method further comprises, prior to step (d), adding the biologically active agent to the mixture.

In a ninth aspect, the invention features a method for preparing a polymerized hydrogel comprising the steps of: (a) combining a hydrophilic macromer and a hydrophobic, water-insoluble macromer; (b) heating and stirring the junction formed in step (a) to form a homogeneous mixture; (c) cooling the mixture to room temperature; (d) adding water and an initiator to the mixture and allowing the mixture to swell; and (e) polymerizing the macromer and forming a hydrogel. Preferably, the method further comprises, prior to step (e), adding the biologically active agent to the mixture.

In a tenth aspect, the invention features a method of protein delivery comprising the steps of: (a) combining a protein with a polymerizable hydrophilic polymer; (b) forming a mixture of the combinations formed in step (a); (c) polymerizing the mixture to form preparations; and (d) administering the compositions or a portion thereof to a mammal, wherein the protein remains intact and wherein at least 70% of the protein is released from the compositions.

In an eleventh aspect, the invention features a method of delivering a biologically active agent, the method comprising the steps of: (a) combining the active agent with a biodegradable, polymerizable macromer in aqueous solution in the presence of a free radical initiator; (b) dispersing the solution and forming fine droplets containing the macromer and the biologically active agent; (c) polymerizing the macromer in droplets to form hydrogel particles having a biologically active agent incorporated therein, wherein the particles are capable of controlled release of the biologically active agent; and (d) administering the compositions or a portion thereof to the mammal, wherein step (c) proceeds in the absence of vinylpyrrolidone monomer. Preferably, at least 80% of the particles have a particle size of less than 5 µm.

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A twelfth invention features a composition comprising a biologically active agent enclosed within a biodegradable, polymerizable macromer, wherein the macromer comprises at least one water-soluble region, at least one degradable region that is hydrolyzable under n / v conditions, and polymerizable end groups capable of forming further covalent bonds resulting in polymerization of the macromer, wherein the polymerizable end groups are separated by at least one degradable region, and the composition comprises at least 5 weight percent of the active agent.

A thirteenth invention is characterized by an insoluble macromer comprising at least one water-soluble region, at least one degradable region that is hydrolyzable under in vivo conditions and polymerizable end groups having the ability to form additional covalent bonds resulting in polymerization of the macromer, wherein the polymerizable end groups are separated by at least one degradable region.

Fourteen, the invention features a sustained-delivery protein composition, wherein the composition comprises an insoluble macromer with at least one water-soluble region, at least one degradable region that is hydrolyzable under in vivo conditions, and polymerizable end groups that have the ability to form additional covalent bonds having as a result of macromer polymerization, wherein the polymerizable end groups are separated by at least one degradable region.

In a fifteenth aspect, the invention features a macromer comprising at least one water soluble region, at least one degradable region that is hydrolyzable under in vivo conditions, and polymerizable end groups that have the ability to form additional covalent bonds resulting in polymerization of the macromer, wherein the polymerizable end groups are separated at least one degradable region and the degradable region consists essentially of polytrimethylene carbonate.

In a sixteenth aspect, the invention features a composition for subcutaneous administration of LHRH, wherein the composition comprises a polyethylene glycol core having a molecular weight of about 1000 daltons and a degradable region consisting of polycaprolactone, wherein the composition is capable of administering a therapeutic dose of LHRH for more than 30 days.

In a seventeenth invention, a composition comprising a glucagon-like peptide-1 and a macromer comprising at least one water-soluble region, at least one degradable region that is hydrolyzable under in vivo conditions, and polymerizable end groups having the ability to form additional covalent bonds having as a result of macromer polymerization, wherein the polymerizable end groups are separated by at least one degradable region.

In an eighteenth invention, the sustained release hydrogel composition is characterized in that the composition comprises particles with a density of less than 0.4 g / cm ³ , at least 50% of the particles have a mean diameter of less than 5 µm, and wherein the composition is developed for pulmonary administration.

In a nineteenth invention, the sustained release composition of a biologically active agent comprises particles having a density of greater than 0.4 g / cm ³ .

In view of the features of the invention described above, the following embodiment is preferred. The time for which 10% of the releasable active substance is released is greater than 1/10 tso. The macromer preparations and mixtures contain at least 2.5% by weight of the active ingredient and preferably contain at least 5, 10, 25 or 40% by weight of the active ingredient. The macromers comprise: (a) a water-soluble region forming the central core; (b) at least two polymerizable end groups, wherein the polymerizable end groups are attached to degradable regions.

The water-soluble region comprises a polymer selected from the group consisting of polyethylene glycol, polyethylene oxide, polyvinyl alcohol, polyvinylpyrrolidone, polyethyloxazoline, polyethylene oxide and polypropylene oxide copolymers, polysaccharides, carbohydrates, proteins, and combinations thereof. The water-soluble region may comprise at least two arms.

The degradable region comprises a polymer selected from the group consisting of poly-ahydroxy acids, polylactones, polyamino acids, polyanhydrins, polyorthoesters and polyphosphoesters. For example, the degradable region may comprise polytrimethylene carbonate or polycaprolactone. Optionally, the degradable region may comprise a poly-α-hydroxy acid selected from the group consisting of polyglycolic acid, poly-DL-lactic acid, and β-L-lactic acid. The degradable region may optionally contain a polylactone selected from the group consisting of poly-ε-caprolactone, poly-δ-valerolactone and poly-γ-butyrolactone. The degradable region may comprise a copolymer of at least two different monomers or a mixture of at least two different monomers.

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The polymerizable end groups contain a double bond between two carbon atoms, which is capable of causing macromer polymerization.

The compositions are administered to the lungs of a mammal. Alternatively, the formulations are administered intravenously, subcutaneously, intramuscularly, orally or nasally. Preferably, the compositions are administered to humans and the biologically active agent is preferably a protein.

A "therapeutic dose", when a biologically active substance, is its plasma level between the minimum effective level and a toxic level.

"Release kinetics" means the rate at which a drug is released from its dosage form.

'Macromer' means a polymer consisting of three components: (1) a biocompatible, water-soluble region; (2) a biodegradable / hydrolyzable region; and (3) at least two polymerizable regions.

The term "intact" when used in connection with a protein or peptide means that the protein or peptide is in its biologically active form and is not degraded or aggregated.

The terms "water insoluble" or "water insoluble" mean that the solubility of the compound is less than 1 g / 100 mL of an aqueous solution or in an aqueous solution containing up to 5% organic solvent such as dimethylsulfoxide.

The methods and compositions of the present invention allow the controlled release of large amounts of biologically active agents such as proteins. The macromers used to deliver the proteins both protect the proteins from degradation and also allow the rate of release of the proteins to be adjusted. Proteins can be delivered over a period of hours or months. Further, the methods and compositions of the present invention allow a relatively constant dosage of the active agent rather than its sudden initial release.

The invention provides methods and compositions for administering biologically active agents. These methods and compositions allow for the controlled, sustained release of relatively large amounts of these substances.

In one embodiment, the biologically active agent is associated with a biodegradable, polymerizable macromer in the presence of a polymerization initiator. The macromer is polymerized to form a hydrogel and to incorporate the substance into the resulting hydrogel. Preparations are prepared from the resulting hydrogel containing the active ingredient which are capable of controlling the release of the active ingredient.

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Makromery.

The macromers of the present invention have at least one water-soluble region, at least one biodegradable (e.g., hydrolyzable) region, and at least one polymerizable region. The macromers may be water-soluble or water-insoluble. These macromers are polymerized to form hydrogels that are suitable for delivering embedded substances at a controlled rate. An important feature of hydrogels is that the polymerizable regions are separated by at least one biodegradable region. This separation facilitates uniform degradation in vivo.

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

There are several variants of these macromers. For example, the polymerizable regions may be attached directly to the degradable regions; optionally, they may be attached indirectly by means of water-soluble, non-degradable regions, wherein the polymerizable region is separated by a degradable region. For example, if the macromer comprises one water-soluble region associated with a degradable region, one polymerizable region may be attached to a water-soluble region and the other to a degradable region.

In another embodiment, the water-soluble region forms the central core of the macromer and has at least two polymerizable regions attached thereto. At least two polymerizable regions are attached to the degradable regions such that after degradation, the polymerizable regions, especially in the form of a polymerized gel, are separated from each other. Alternatively, if the central core of the macromer is formed by a degradable region, at least two water-soluble regions may be attached to the core and the polymerizable regions may be attached to each water-soluble region.

In yet another embodiment, the macromer has a water-soluble backbone, wherein the degradable region is attached to the macromer backbone. At least two polymerizable regions are attached to the degradable regions so that they are separated upon degradation resulting in gel dissolution. In another embodiment, the macromer backbone comprises a degradable backbone having water-soluble regions

attached as protrusions or grafts on a degradable skeleton. Two or more polymerizable regions are attached to these water-soluble processes or grafts.

In another embodiment, the carcass may have a plurality of side arms; for example, it may be star-shaped or comb-shaped. The skeleton may comprise a water-soluble region, a biodegradable region, or a water-soluble, biodegradable region. The polymerizable regions are attached to this framework. Again, here, the polymerizable regions must be separated at some point by the degradable region.

Throughout this description, the following abbreviations are sometimes used to describe the specific macromers of the present invention. In two particular examples, a macromer whose water-soluble region consists of polyethylene glycol having a molecular weight of 4000 daltons, with five lactate groups on each side of the region and bounded on each side by acrylate groups, is referred to as "4KL5". Similarly, a macromer whose water-soluble region consists of polyethylene glycol with a molecular weight of 3400 Daltons, with six caprolactone groups on each side of this region and bounded on each side by acrylate groups, referred to as "3.4KC6".

Water-soluble areas

The water-soluble region may comprise polyethylene glycol, polyethylene oxide, polyvinyl alcohol, polyvinylpyrrolidone, polyethyloxazoline, copolymers of polyethylene oxide and polypropylene oxide, polysaccharides, carbohydrates or proteins or combinations thereof.

Preferably, the macromer comprises a water-soluble central region containing polyethylene glycol (PEG) because PEG is highly hydrophilic and water-soluble, and also has high biocompatibility. Preferably, the polyethylene glycol region has a molecular weight of 400 to 40,000 daltons, and more preferably has a molecular weight of 1000 to 30,000 daltons, 1000 to 20,000 daltons, or 2000 to 10,000 daltons.

Degradable area.

The degradable region may comprise, for example, poly-α-hydroxy acids, polylactones, polyamino acids, polyanhydrins, polyorthoesters, polyorthocarbonates or polyphosphoesters, or mixtures or copolymers of these polymers.

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For example, poly-α-hydroxy acids include polyglycolic acid, poly-DL-lactic acid, and poly-L-lactic acid. For example, polylactones include poly-ε-caprolactone, poly-δ-valerolactone, poly-γ-butyrolactone, poly-1,5-dioxepan-2-one, and polytrimethylene carbonate.

Examples of copolymers include a copolymer of caprolactone and glycolic acid; and a caprolactone-lactic acid copolymer.

Polymerizable regions.

The polymerizable regions preferably contain a double bond between two carbon atoms which is capable of causing macromer polymerization. Choosing a suitable polymerizable group will allow rapid polymerization and gel formation. Preferred are the polymerizable regions containing acrylates, as they can be polymerized using a variety of initiation systems, as discussed below. Examples of acrylates are acrylate, methacrylate and methyl methacrylate.

Polymerization step.

Macromers are polymerized by polymerization initiators with the aid of long-wave ultraviolet light, visible light, thermal energy, or a redox oxidation system. The polymerization may be carried out at room temperature or at lower temperatures, for example temperatures below 20 ° C. During polymerization, substances such as proteins are physically incorporated into the resulting polymer gel network.

The polymerization can 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 variety of suitable dyes, such as xanthine dyes, acridine dyes, thiazine dyes, phenazine dyes, camphroquinone dyes, acetophenone dyes, or eosin dyes with triethanolamine, 2,2-dimethyl-2- phenyl acetophenone and 2,2-methoxy-2-phenylacetophenone.

Polymerization can also occur in the absence of light. For example, the polymerization may be initiated by a reduction oxidation system, as described in more detail in the examples. In some cases, it is preferable to polymerize with the reduction oxidation system of the present invention since the formation of radical initiators takes place at a suitable rate over a wide temperature range.

Initiators which can be used in the redox system are, inter alia, peroxides such as acetyl, benzoyl, cumyl and t-butyl; hydroperoxides such as t-butyl and cumyl, peroxoesters such as t-butyl perbenzoate; acylalkylsulfonyl peroxides, dialkyl peroxydicarbonates, diperoxoketals, ketone peroxides, and azo compounds such as 2,2'azo (bis) isobutyronitryl (AIBN), disulfides and tetrazenes.

Properties of macromers.

The objects of the invention are biodegradable. Biodegradation occurs in bonds within the side oligomers, resulting in fragments that are nontoxic and readily removable from the body, or are normal, safe chemical intermediates found in the body. These materials are particularly useful for delivering hydrophilic materials since the water-soluble regions of the polymer allow water access to the material trapped within the polymer.

More importantly, the subject invention can be degraded under in vivo conditions at rates that allow controlled release of the incorporated substances. Release may occur by diffusion of the polymer material prior to degradation or by diffusion of the polymer material during degradation. Polymer degradation facilitates the final controlled release of free macromolecules in vivo by sequential hydrolysis of terminal ester bonds. Already in the field of preparation, this avoids the rapid release of the biologically active substance at the beginning of administration, which is sometimes associated with other release systems.

The release rate depends in part on the composition of the water-soluble region, such as the molecular weight of the components of the water-soluble region. The release rate of a biologically active agent may also depend on the degree of polymerization of the macromer as well as other factors.

The rate of substance release also depends on the degradation rate of the degradable region of the macromer. For example, glycolic acid esters result in very rapid degradation, lactic acid esters result in somewhat slower degradation, and caprolactam esters result in very slow degradation. If the degradable region consists of polyglycolic acid, the release period is less than one week. If the degradable region consists of polylactic acid, the release period is about one week. If the degradable region consists of caprolactone-lactic acid copolymers or trimethylene carbonate-lactic acid copolymers, the release period is two to four weeks. If the degradable region consists of trimethylene carbonate copolymers · · ··· · · · · · · · · ♦ · ♦ · φ · φφ φφφφφφ olymerφφolymerφolymerφφolymerolymerolymerolymerolymerolymerolymerolymerolymerolymer a kap a trimethylene carbonate, the release period is three to eight weeks. If the degradable region consists of trimethylene carbonate or polycaprolactone, the release period is longer than five weeks.

The exact release rate can be further modified by varying the ratio of hydrophilic and hydrophobic components. For example, a highly soluble macromer will yield a hydrophilic gel after polymerization; it has been shown that hydrophilic hydrogels are degraded more rapidly than hydrophobic hydrogels. A mixture of a hydrophilic macromer (eg 4KL5) with a hydrophobic, water-insoluble macromer (3.4KC6) is used to prepare polymerized hydrogels. The hydrogel will have a release rate that is between the release rate of lactic acid-only hydrogels and caprolactone-only hydrogels. The macromer in which the degradable region is a caprolactone-lactic acid copolymer will also have a release rate that is between the release rate of lactic acid-only hydrogels and caprolactone-only hydrogels as the primary degradable groups.

Further, the rate of release from a given article depends on the amount of incorporated substance, its percentage in the final product; the solubility of the active agent; on hydrophilicity of the active substance (hydrophilic active substances will generally be released more rapidly than hydrophobic active substances); and, in the case of suspensions, to particle sizes. By adjusting the factors discussed above, degradation and controlled release can be varied over very wide ranges. For example, the formulation may be designed to release over hours, days, or months.

As shown in Fig. 1, particles that behave as homogeneous drug delivery systems can be formed by the methods of the present invention. Due to the homogeneous nature of the objects of the present invention, there is no initial sharp increase in the concentration of the released substance. Furthermore, uniform consistency allows for the incorporation of relatively high amounts of protein, while still minimizing the initial sharp increase in the concentration of the released substance.

In general, water-soluble substances will form homogeneous systems when incorporated into the macromers of the present invention. Substances that do not dissolve in water during the time of formation of the macromer of the present invention will form heterogeneous systems. The extent of a steep initial increase in the concentration of the released substance in heterogeneous systems can be minimized by a special small particle suspension.

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The course of substance release is shown in Fig. 2. The horizontal axis shows the time after administration and the vertical axis represents the amount of material released. As shown in Fig. 2, the time t ₅₀ is the time at which 50% of the releasable material has been released. Similarly, the time tw is the time at which 10% of the releasable material has been released. The amount of releasable active substance is the amount that is released from the formulation over a period of ten times longer than the time it takes to release 10% of the incorporated active substance.

If the release curve is exactly linear then tw = 1/5 tso- If there is an initial sharp increase in the concentration of the released substance, then t _{0 0} is much smaller than 1/5 tso- In the methods and compositions of the present invention, it is preferred that t ₁₀ is greater than 1/10 t ₅ o. In other words, there is no or very small initial increase in the concentration of the released substance.

The invention also features insoluble macromers. These macromers comprise at least one water-soluble region, at least one degradable (eg, hydrolyzable) region, and at least one polymerizable region. The degradable region comprises polymers of glycolic acid, lactic acid or caprolactone, trimethylene carbonate or mixtures or copolymers thereof. The degradable area must be insoluble in water. For example, a macromer may be prepared having a degradable region comprising 15 to 20 lactic acid units; this macromer will allow a relatively high release rate. A degradable region macromer containing 6 caprolactone units will allow a relatively slow release rate. A degradable region macromer comprising a copolymer of six units of caprolactone, four units of lactic acid and four units of glycolate will allow high release rates, and a degradable region macromer comprising a copolymer of three units of lactic acid and seven units of trimethylene carbonate will allow medium release rates.

The water-soluble region of these macromers is preferably PEG. The water-soluble region can have many arms; for example, it may be star-shaped or comb-shaped. The water-soluble region preferably has 4, 6 or 8 arms and a molecular weight of 10,000 to 40,000 daltons.

Characteristics of the amount of incorporated substance.

Therapeutic agents can be readily incorporated with great yield into the compositions described herein. For example, preparations can be made containing at least 2.5

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9 9 9 · 9% by weight of the active substance. It is preferred that the compositions contain at least 5, 10, 25 or 40 weight percent of the active ingredient.

The amount of incorporated active agent can be measured by dissolving the formulation pieces in a suitable solvent and measuring the amount of active agent using methods available in the art, such as spectrophotometry.

Shaping products.

The formulations prepared by the procedures described above may be formed into any desired shape. For example, the formulations may be shaped to conform to a particular body cavity. They can also be formed into thin, flat discs or microspheres. Another possibility is that the formulations can be preformed and then adapted to the desired shape only before use or ground to fine particles. The desired shape of the formulation will depend on the specific use.

The particles can be prepared using techniques known in the art, including simple or repeated evaporation of the solvent from the emulsion, spray drying, and solvent extraction. The term "particles" as used herein includes, but is not limited to, microspheres. In a microsphere, the therapeutic or other agent is substantially dispersed throughout the volume of the particle. The particles may have a smooth or irregular surface and may be solid or porous. Methods for forming microspheres are described in the literature, for example in Mathiowitz and Langer, J. Controlled Release 5: 13-22 (1987); Mathiowitz et al., Reactive Polymers 6: 275-283 (1988); Mathiowitz et al., J. Appl. Polymer Sci. 35: 755-774 (1988); Mathiowitz et al., Scanning Microscopy 4: 329340 (1990); ); Mathiowitz et al., J. Appl. Polymer Sci. 45: 125-134 (1992); and Benita et al., J. Pharm. Sci. 73; 1721-1724 (1984).

Upon evaporation of the solvent described, for example, in Mathiowitz et al., (1990), Benita et al., (1984) and US Patent No. 4,272,398, the polymer is dissolved in a volatile organic solvent such as methylene chloride. Optionally, the agent to be incorporated is added to the polymer solution either in a soluble form or dispersed as fine particles, and the mixture is suspended in an aqueous phase containing a surfactant such as polyvinyl alcohol. The resulting emulsion is stirred until most of the organic solvent has evaporated to leave solid microspheres which can be washed with water and dried overnight in a lyophilizer.

• 4,444

Upon removal of the solvent, the therapeutic or diagnostic agent is dispersed or dissolved in a solution of the selected polymer in a volatile organic solvent such as methylene chloride. The mixture can then be dispersed in an oil such as silicone oil by stirring until an emulsion is formed. As the solvent diffuses into the oil phase, the emulsion droplets solidify into solid polymer microspheres.

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

Spray drying is accomplished by passing a homogeneous mixture of a substance, such as a therapeutic agent and a polymerizable macromer, used to form a hydrogel, through a nozzle, a rotary disk or an analogous device to spray the mixture in the form of fine droplets. The agent and the polymerizable macromer may be provided in a solution or suspension, such as an aqueous solution. The fine droplets are exposed to light which causes the polymerization of the macromer and the formation of hydrogel droplets containing the substance in question.

In another embodiment, the hydrogel particles are prepared using an oil-in-water emulsion wherein the polymerizable macromer and the substance to be incorporated are dispersed in the water-in-oil emulsion and exposed to light to polymerize the macromers and form hydrogel particles containing the substance, such as a biologically active agent. . Typically, the polymerization can be carried out at room temperature.

The microspheres prepared using the techniques described above are freeze-dried so that they have a long shelf life (without biodegradation) and the drug remains biologically active. Before use in an injectable form, the microspheres are diluted in a suitable solution, such as physiological saline or other liquids. For pulmonary administration, freeze-dried or diluted particles may be used.

Biologically active substances.

Biologically active agents that can be incorporated into the compositions of the present invention include therapeutic, diagnostic and prophylactic agents. They may be naturally occurring compounds, synthetic organic compounds or inorganic compounds. Substances that can be incorporated into the compositions of the present invention include proteins, peptides, sugars, inorganic agents, antibiotics, antineoplastic agents, local anesthetics, anti-angiogenic agents, vascular agents, anticoagulants, immunomodulators, cytotoxic agents, antiviral agents 99 agents, antibodies, neurotransmitters, psychoactive drugs, oligonucleotides, lipids, cells, tissues, tissue or cell aggregates and combinations thereof.

Examples of therapeutic agents include calcitonin, granulocyte macrophage colony stimulating factor (GMCSF), ciliary neurotrophic factor, parathyroid hormone, and the transmembrane regulatory cystic fibrosis gene. Other specific therapeutic agents include parathyroid hormone-like peptide, somatostatin, testosterone, progesterone, estradiol, nicotine, fentanyl, norethisterone, "clonidine", scopolamine, salicylate, salmeterol, formeterol, albeterol and valium.

Medicines can be used to treat pneumonia, including pentamidine isethiouate. Medicines for the treatment of pulmonary conditions such as asthma may be used including albuterol sulphate, βagonists, metaproterenol sulphate, beclomethasone diprepionate, triamcinolone acetamide, budesonide acetonide, ipratropium bromide, flunisolide, cromolyn sodium sodium, wine ergotamine and protein or peptide drugs such as TFN antagonists or interleukin antagonists.

Other therapeutic agents include cancer chemotherapeutic agents such as cytokines, lymphokines, DNA, and vaccines such as attenuated influenza virus. Nucleic acids that can be incorporated include genes, cDNAs encoding proteins, expression vectors, molecules having positive polarity that bind to complementary nucleic acid sequences to inhibit transcription or translation, and ribozymes. For example, genes may be administered to treat diseases such as cystic fibrosis. Polysaccharides such as heparin may also be administered.

Other therapeutic agents include tissue plasminogen activator (t-Pa), superoxide dismutase, luteinizing hormone releasing hormone (LHRH) catalase antagonists, platelet factor IL-11, IL-4 receptor, "enbrel", IL-2 receptor antagonists, TFN fusion proteins receptor, megakaryocyte growth and development factor (MGDF), stem, anti-HER-2 and anti VEGF humanized monoclonal antibodies, antiTac antibodies, GLP-1 amylin, and GLP-1 amylin analogs.

Other therapeutic agents include atrial natriuretic factor, atrial natriuretic peptide, human chorionic β-gonadotropin, basic fibroblast growth factor, bovine growth hormone, bone morphogenetic protein, B-cell stimulation factor-1, B-cell stimulation factor-2, bovine somatotropin, Carcinobreaking factor, cartilage inducing factor, corticotropin releasing factor, colony stimulating factor, differentiation factor-1, endothelial cell growth factor, erythrocyte differentiation factor, • fe • fefefe • fe elongation factor 1-alpha, epidermal growth factor, erythropoietin, growth factor fibroblast factor, follicle stimulating hormone, granulocyte colony stimulating factor, glial fibrallary acid protein, growth hormone releasing factor, human alpha-1 antitrypsin, human atrial natriuretic factor, human chorionic gonadotropin, human growth hormone, human le inhibiting factor haemopoietin-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-11, immunoglobulin G , immunoglobulin M, interleukin-1, interleukin-2, interleukin-3, interleukin-4, interleukin-5, interleukin-6, renal plasminogen activator, cell adhesion lectin molecule, luteinizing hormone, leukemia inhibiting factor, monoclonal antibody, activating factor macrophages, cytotoxic factor for macrophages, macrophage colony stimulating factor, megakaryocyte colony stimulating factor, tumor necrosis factor A, macrophage inhibiting factor, Mullerian inhibiting agent, megakaryocyte stimulating factor, melanocyte stimulating factor, neutrophil chemotactic factor, neural growth factor activator, neural growth factor, activator nester oid anti-inflammatory drug, osteogenic extract factor, antitumor lymphokine, prostate-specific antigen, plasminogen activator inhibitor, platelet-derived growth factor, platelet-derived wound healing agent, human interleukin-inducing plasma protein, tumor vascular formation factor, tissue control factor, T cell growth factor, T cell modulating peptide, transforming growth factor, tumor growth inhibitor, tumor inhibiting factor, tissue metalloproteinase inhibitor, tissue necrotic factor, tissue plasminogen activator, vascular endothelial growth factor, and vasoactive intestinal peptide.

Examples of diagnostic agents include gases and other commercially available imaging agents that are used in positron emission tomography (PET), computed tomography (CAT), photon emission computed tomography, X-ray, fluoroscopy, and magnetic resonance imaging (MRI). Suitable materials used as contrast agents in MRI include gadolinium chelates as well as iron, magnesium, manganese, copper and chromium chelates. Examples of materials suitable for CAT and X-ray include iodine-based materials.

A preferred biologically active agent is a protein. Proteins are defined as containing 100 or more amino acid residues; the peptides contain less than 100 amino acid residues. Unless otherwise indicated, the term protein refers to both the tttt proteins and peptides of tttt ttt ttt tttt ttt ttt proteins and peptides. Proteins can be prepared, for example, by isolation from natural sources or by recombinant means. Examples include insulin and other hormones, including growth hormones such as human growth hormone and bovine growth hormone. Other examples of proteins include Factor VIII, Factor IX, Factor VIIa, and anti-inflammatory agents such as interleukins, including interleukin-4, NSAIDs or corticosteroids. Other examples of proteins include enzymes such as DNase and proteases. Other proteins include cytokines, interferons including interferon alpha and interferon beta, poetins, colony stimulating factors, growth factors, ceredase, gibberellins, auxins and vitamins and fragments thereof. Examples of growth factors include vascular endothelial growth factor (VEGF), epithelial cell growth factor (ECGF), fibroblast growth factor (bFGF), and platelet-derived growth factor (PDGF).

Proteins are stable in the hydrogels of the present invention. For example, many proteins are protected from dimerization or aggregation, as discussed further in the examples. Enzymatic degradation of proteins or peptides can be further minimized by the simultaneous incorporation of peptidase inhibitors.

Routes of administration.

Inhalation.

The use of the hydrogel particles of the present invention can extend the delivery of drugs to the lungs. Administration to the lung allows delivery of drugs that can be transported across the lung tissue barriers and into the circulation as described in US Provisional Patent Application Serial No. 60 / 053,029, filed July 18, 1997.

The problem with the delivery of active substances to the lungs is that pulmonary macrophages can absorb the substances and prevent them from entering systemic or local circulation. Absorption occurs when proteins adsorbed on the surface of the particle bind to receptors on the surface of macrophages. In order to prevent absorption, the invention provides nonionic hydrogels, e.g. consisting of polymers based on polyethylene glycol. These hydrogels adsorb small amounts of proteins and thus weakly bind to cell surfaces. Anionic hydrogels, e.g. formed with polyacrylic acid, also adsorb relatively small amounts of proteins and thus weakly bind to cell surfaces.

In another embodiment, biocompatible microspheres can be formed and their surface provided with non-ionic water-soluble polymers such as polyethylene oxide (PEO) to create resistance to cell adhesion as described in US Patent No. 5,380,536.

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The size and density of the particles can also be selected to maximize the amount of active agent that is delivered to the lungs. For example, macrophages do not absorb large particles as effectively as small particles. However, large particles are not delivered as deep into the lungs as small particles are. To overcome these contradictory factors, the invention provides small particles that can swell as they hydrate. The particles are administered deep into the lungs as small (i.e. 1 to 5 µm), dry or slightly moist particles; due to hydration they swell and thus become resistant to absorption by pulmonary macrophages. Swelling can occur when particles are hydrated from a dry state and when hydrated from one state of hydration to another by changing the temperature, pH, salt concentration, or presence of other solvents, for example depending on the chemical and physical nature of the hydrogel polymer.

As used herein, the term "dry" means that the powder particles have a moisture content such that the powder is easily dispersed by the inhalation device and forms an aerosol. It is preferred that the moisture content of the particles is less than 10 weight percent, more preferably less than 5 weight percent or optionally less than 2 weight percent or less.

The density of the particles is determined by measuring the density of the deposited powder. The density of the deposited powder is a standard measure for the density of the mass in a volume bounded by a spatial area. The density of matter in the volume bounded by the spatial surface of an isotropic particle is defined as the mass of the particle divided by the volume of the minimum spherical surface within which the particle can be enclosed. Particle density can be measured using GeoPyc (Micrometers Instrument Corp., Norcross, GA) or AutoTap (Quantachrome Corp., Boyton Beach, FL).

For example, the particle density of 3.4KL5 was determined as follows. 3.4KL5 (1.0025 g), 200 mM TEOA in PBS pH 7 (1.0260 g) and 1000 ppm eosin were mixed. 200 mg of this solution was mixed with talc (0.1015 g). The resulting suspension was placed in a 100 μΙ glass pipette and polymerized with light for 15 s (ILC Technology, Inc., Xenon fiber optic light source). The rod was extruded, placed on an aluminum foil, and further polymerized for 3.5 min. The solid bar was lyophilized (vacuum 15E-3 mbar, temperature -50 ° C) for 18 h. The dry bar (water content <10%) was cut into small pieces, placed in heptane and ground into small particles in a homogenizer ( Silverson L4RT-A) at 5,000 rpm. (rpm). The wet particles were air dried, followed by a stream of nitrogen. The particle size ranged from 1 µm to 0.5 mm.

1.645 g of these particles were placed in a 10 ml graduated cylinder. The graduated cylinder was mounted on top of an AutoTap densitometer (Quantachrome). The sample was shaken 100 times and the particle volume was read. The procedure was repeated until no changes in volume were observed. The final volume was 2.8 ml. The particle density of the deposited powder was 1.6435 g / 2.8 mL = 0.5870 g / mL.

In addition to the particles, the polymer may be provided in other shapes suitable for delivery deep into the lung. For example, PEG emulsion microspheres are subjected to high pressure and vacuum on a flat plate to form very light and very thin slices, for example having a snowflake consistency, which respond differently to forces in the air stream. For example, the resulting thin flakes may be 0.01 µm, 1 µm or 10 µm thick.

The particles may be administered to the respiratory system alone or in any suitable, pharmaceutically acceptable base, such as a liquid, eg saline, or powder. Aerosol dispensing, formulations, and delivery systems can be selected for particular therapeutic applications, as described, for example, in Gonda, I. "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 Mořen, "Aerosol dosage forms and formulations" in: Aerosols in Medicine. Principles, Diagnosis and Therapy, Mořen et al., Eds. Elsevier, Amsterdam, 1985.

Drug delivery to the lung can be achieved by devices such as fluid nebulizers, aerosol metered dose inhalers and dry powder dispersing devices. For use in dry powder dispersion devices, the polymer particles containing the therapeutic agent are prepared in the form of a dry powder, for example by lyophilization or by drying the sprayed material. Methods using drying the sprayed material to prepare dry powders for pharmaceutical use comprising a pharmaceutically acceptable amount of a therapeutic agent and a carrier are described in PCT WO 96/32149.

Examples of drugs that can be administered to the lung include, but are not limited to, insulin, antitrypsin, calcitonin, alpha interferon, beta interferon, GLP-1, and DNase.

Nasal administration.

..............

The compositions may also be used for nasal administration of the compounds. For example, a vaccine containing freeze-dried or diluted microspheres may be administered nasally.

Intramuscular and subcutaneous administration.

The objects of the present invention can be used to administer microspheres that are degraded over several days to 3 months by intramuscular injection or subcutaneous injection.

For example, growth hormone may be administered subcutaneously; the hormone leaves the microspheres at the injection site where they are degraded. Growth hormone enters the systemic circulation, where it exerts its effects directly and at the same time indirectly due to the induction of somatomedin production in the liver.

Particles up to 0.5 mm in size can be used for this type of administration.

In other embodiments, the active agent is a vaccine, such as a tetanus vaccine, other peptides or proteins, or more complex immunogens. The vaccine is released over a period of time from one to many weeks, resulting in, at the same total immunogen dose, an improved immune response to the vaccine as compared to a primary injection followed by one or more booster doses. Mixtures of different types of microspheres may also have results in a basic injection followed by booster doses.

Intravenous administration.

Hydrogel microspheres containing a drug suitable for the treatment of blood clotting disorders such as Factor VIII or Factor IX in haemophilia may be administered by intravenous injection. The drug is released over a period of days to weeks. A therapeutic drug level that maintains a better clinical outcome is maintained. Furthermore, it is possible to administer generally lower doses of the drug, which has a correspondingly favorable economic effect. These approaches help to induce patient cooperation.

In the case of intravenous injection, it is important to make the microspheres an acceptable product, that is, the microspheres do not aggregate and clog the blood vessels. The microspheres must be prepared in a suitable size so that they do not get stuck in capillaries. For this type of administration, particle sizes of 0.2 to 0.5 µm are preferred.

Part of a number of inflammatory conditions, where part of the inflammatory process is due to selectin and the expression and binding of ICAM to neutrophil intravisation, is that blood vessels become permeable at the site of inflammation. Hydrogel microspheres may be administered; these microspheres escape from the blood vessels at the site of inflammation and then release their drug content for a period of time. Diseases in which this approach may be useful may include, but are not limited to, inflammatory bowel disease, asthma, rheumatoid arthritis, osteoarthritis, emphysema, and cystic fibrosis (with DNase as an enzyme drug).

Hydrogel microspheres that contain cytokines, lymphokines or other compounds for treating cancer can be administered by intravenous injection. Blood vessels inside large, solid tumors are commonly permeable and blood flow through them is often slow. Thus, microspheres can settle inside solid tumors and release their anticancer drugs locally, killing the tumor cells directly or by local activation of the immune system. This approach can be used, for example, with compounds such as interleukin 2, where systemic and local toxicity limited dose levels and where there were significant side effects.

The microspheres of the present invention will be relatively slowly removed from the circulation. Alternatively, the microspheres may be pre-targeted to exit the circulatory system through permeable blood vessels or through more active target mechanisms, eg, a receptor-mediated target mechanism.

Oral administration.

In some parts of the gastrointestinal tract, there is a relatively good transport of proteins across the intestinal mucosa into the systemic and local circulation. The compositions of the present invention, for example, freeze-dried microspheres containing proteins (very small particle sizes), can therefore be administered orally in forms suitable for the gastrointestinal tract that protect microspheres containing drugs against attack by enzymes and low pH found in in the upper part of the digestive tract. Such forms suitable for the gastrointestinal tract can also be designed using some of the available technologies to gradually eliminate drug-containing microspheres from the gastrointestinal enteric capsule process. This is described in more detail in the provisional application of USSN 60/053 029 and in Mathiowitz et al., Nature 386 (6623): 410414 (1997). This approach is believed to have a number of advantages over other approaches for delivering proteins and other molecules, even small molecules, by the oral route. First, PEG and proteins are compatible with each other so that most manufacturing and stability problems encountered in other drug delivery approaches can be avoided. Second, the dried hydrogels are very adhesive to wet tissue. The microspheres bind well to the gastrointestinal tract and will be transported to the system via the digestive tract circulation or release their contents on the intestinal mucosa; however, the drug enters the systemic circulation and the digestive tract. Chemical enhancers or forms containing compositions that utilize specific and non-specific biological transport mechanisms to facilitate transport through the digestive tract to the systemic circulation may also be included.

Targeted action.

Target ligands can be attached to the particles by means of reactive functional groups found thereon. Target ligands allow binding interactions of the particle with specific receptor sites, such as found within the lung or on mucosal cells, specific for different regions of the vascular bed. Target ligands are selected according to specific or non-specific possibilities for their binding to certain targets. Examples of target ligands include antibodies and fragments thereof, including antibody variable regions, lectins, hormones, or other organic molecules capable of specifically binding to receptors on the surface of target cells. Other ligands are described in Science 279, 323324 (1998).

Microspheres can be prepared with both the drug and the target molecule. It is also possible to make double microspheres wherein the inner sphere contains the drug and the outer shell of PEG comprises the target molecule or reagent.

Ointment bases and carriers.

The particle incorporating the therapeutic or diagnostic agent may be provided in combination with one or more pharmaceutically acceptable excipients available in the art, such as described in PCT WO 95/31479. The ointment bases may be selected such that they can increase stability, dispersibility, density, and volume in some applications to ensure uniform delivery to the lungs. The ointment base can be, for example, human serum albumin (HSA), bulking agents such as sugars, amino acids, peptides, pH adjusters or buffers and salts. Other ointment bases include zinc, ascorbic acid, mannitol, sucrose, trehalose, cyclodextrans, polyethylene glycol, and other commonly used pharmaceutical ointment bases, including those described in the United States Pharmacopeia Convention, Inc., 1995 (cf. eg p. 22052207). Examples of sugars are monosaccharides such as galactose and disaccharides such as lactose. Particularly suitable are ointment bases which stabilize proteins.

• · • ·

In some cases, the ointment bases are used as carriers; i.e., they are used to adjust the release rate of the active agent. For example, mannitol may be used to accelerate or slow the release rate.

The following are specific examples which describe the preparation of the compositions of the present invention and the methods of the present invention. These examples are provided to illustrate the invention and are not to be construed as limiting the invention.

In some of the following examples, a macromer prepared from a acrylate-PLA-PEG-PLA-acrylate triple block copolymer was used. PEG had a molecular weight of 3,400 daltons; polylactic acids on both sides had an average of 5 lactic acid units on each side; they are therefore referred to herein as “3.4KL5”. If PEG with a lower molecular weight such as 2000 daltons was used, the resulting macromer is designated "2KL5".

In other examples, the acrylate-PCL-PEG-PCL-acrylate macromer was used. PEG had a molecular weight of 3,400 daltons and had polycaprolactone on both sides, with an average of 6 caprylic units on each side. The polymer is designated herein as "3.4KC6".

All animal studies described here have been conducted with the approval of the Constitutional Committee for the Welfare and Use of Animals.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is a diagram showing particles in which the protein particles are unevenly dispersed in the carrier environment and particles in which the protein particles are dispersed evenly in the carrier environment.

Figure 2 is a graph showing the release pattern of a substance from a mixture with a macromer.

Figure 3 is a graph showing the course of bST release from a mixture of 3.4KL4 and PEGDA.

Figure 4 is a graph showing the course of insulin release from 3.4KL5.

Figure 5 is a graph showing the daily and cumulative ZnbST release from a 50:50 3.4 5KC6 and 3.4 5KL6 mixture.

Figure 6 is a graph showing the daily and cumulative ZnbST release from a 75:25 3.4KL5 and 3.4KC6 mixture.

• · • ··· · ........ · ’··

Figure 7 is a graph showing the daily release of ZnbST monomer, dimer, and soluble monomer from a mixture of 75:25 3.4KL5 and 3.4KC6.

Figure 8 is a graph showing the effect of bST injections and sustained delivery of bST formulation on the growth of pituitary-depleted rats.

Figure 9 is a graph showing the initial release of bST from cylindrical small and large diameter hydrogel devices.

Figure 10 is a graph showing the effect of EPO injections and sustained delivery of EPO formulation on bST percent reticulocytes.

Figure 11 is a graph showing the effect of subcutaneous insulin injections and sustained subcutaneous release of a hydrogel insulin preparation on blood glucose levels in diabetic rats.

Figure 12 is a graph showing the effect of sustained release of the hydrogel insulin preparation in the lung on blood glucose levels in diabetic rats.

Figure 13 is a graph showing the in vitro release rate of EPO from 3.4KL5.

Figure 14 is a graph showing the in vitro rate of insulin release from 3.4KL5 particles.

DETAILED DESCRIPTION OF THE INVENTION

Example 1: General solution preparation

The protein was weighed and the following ingredients were added: (i) 90 mM TEOA / PBS, pH 8.0; (ii) 35% n-vinylpyrrolidone (n-VP); and (iii) 1000 ppm eosin. The resulting mixture was well mixed with a spatula. The solution was left in the dark for 10 min. or until the macromer has adsorbed all of the solution or until the solution is homogeneous.

A macromer solution with the following components was prepared.

Amount protein Amount 90 mM TEOA Amount 35% n-VP Amount of 1000 ppm eosin Amount 3.4KL5 Amount 2KC6 Overall amount 15 mg 57 mg 15 mg 3 mg 45 mg 0 mg 135 mg 15 mg 57 mg 15 mg 3 mg 0 mg 45 mg 135 mg

Example 2: Preparation of a hydrogel from a water-insoluble macromer · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·

..............

0.5 g of 3.4KC6 was added to a 20 ml scintillation vial. To this was added 0.5 ml of 200 mM TEOA, pH 6.95 in PBS buffer, and the macromer was allowed to swell. The macromer was then stirred until it formed a homogeneous mixture. To this mixture was added 20 µl of a 1000 ppm eosin solution in PBS, 10 µl of a 35% n-VP solution, and 0.0845 g ZnbST.

The resulting solution was placed on a silicone slide. Using 0.4 ± 0.2 mm thick plastic film pieces used as spacer, another silicone glass was placed on top and held in place by clamps.

The light source (ILC Technology, Inc. xenon fiber optic light source) was adjusted by a clamp rack at about 5 cm distance to irradiate the slides with this light source. Both sides of the disc were irradiated for 2 min to form a matte disc.

Example 3: Preparation of hydrogels from a 50:50 mixture of water-soluble and water-insoluble macromer

0.56 g of 3.4KL5 and 0.56 g were placed in a scintillation vial. The vial was placed in an oven at 52 ° C; the mixture was stirred occasionally until it formed a homogeneous mixture. It was then cooled to room temperature. To 0.5 g of the above mixture was added 0.5 mL of 200 mM TEOA and pH 6.95 / PBS buffer. The resulting macromer was allowed to swell.

After swelling, the macromer was stirred until it formed a homogeneous mixture of dough consistency. To this mixture was added 20 μΙ 1000 ppm eosin solution in PBS, 10 μΙ 35% n-VP solution and 0.0845 g ZnbST. The resulting solution was stirred and then placed on a silicone slide. Using 0.4 ± 0.2 mm thick plastic film pieces used as spacer, another silicone glass was placed on top and held in place by clamps.

The light source (ILC Technology, Inc. xenon fiber optic light source) was adjusted by a clamp rack at about 5 cm distance to irradiate the slides with this light source. The center of the disc was irradiated; both sides of the disc were irradiated for 2 min to form a matte disc.

Example 4: Preparation of microspheres using a redox oxidation initiation system

300 mg was dissolved in 1 ml of PBS containing 0.5% ammonium persulfate

3.4KL5. 30 ml of silicone oil (chemically pure) was degassed with nitrogen.

• • • • fe fefefe • fe • ·

..............

To the oil was added 0.25 ml of an aqueous medium containing the macromer and stirred at 2000 rpm. on Silverson homogenizers equipped with a 5/8 diameter head. After the mixture was stirred vigorously for 5 min., 0.5 ml of tetramethylethylenediamine was added. The resulting emulsion was stirred for 30 min. After 30 min. 20 ml of heptane was added. The resulting suspension was centrifuged for 2 min. at 2000 rpm. and removed from the bottom of the centrifuge tube. The resulting microspheres were analyzed using a 400X light microscope using phase contrast. The average microsphere size was found to be 2.5 µm.

Example 5: Long-term release of bST

Preparation of the preparation: A mixture of degradable macromers (3.4KL5) and non-degradable macromers (PEG-diacrylate), molecular weight 3400) was used. The protein used was ZnbST (Monsato / Protiva). Protein was added based on dry weight of 20%. The following 3 samples were prepared.

Sample preparation: 20 µl of a bST-precursor solution was prepared as described in Example 1. The mixture was pipetted with a precision extruder pipette and a siliconized glass tip. The solution was placed on a silicone slide. Using 0.4 ± 0.2 mm thick plastic film pieces used as spacer, another silicone glass was placed on top and held in place by clamps. The light source (ILC Technology, Inc., Xenon fiber optic light source) was adjusted with a clamp rack at about 5 cm distance to irradiate the slides with this light source. The center of the disc was irradiated; each side of the disc was irradiated for 2 min.

The clamps, slides, and spacer were carefully removed. Using a spatula and tweezers, the discs were removed and weighed on a clean, tared, silicone slide. The discs were placed in a heat sealable membrane bag as described in more detail below. One 20 µl disc was placed in each bag. The bag was heat sealed, placed in 2.0 mL release media in PBS (0.01% NaN ₃ , 0.05 M PBS, pH 7.4), placed on a shaker at 100 rpm. and incubated at 39 ° C.

At each time point examined, the bag was placed in fresh 2.0 mL PBS release medium. Samples were taken for analysis every day for as long as bST was released.

Tt tt

.......... ·· ·

Membrane bags were prepared as follows. The membrane sheets were cut into pieces of approximately 7 x 2.5 cm. The leaves were folded in half. Using a Bunsen burner or propane burner, the spatula was heated to red heat. The edges of the sheets were flush and the edge of the membrane was cut red with red-hot tweezers to seal the sides. Once the disc was placed in the bag, the last page was sealed using the same procedure.

Samples were analyzed daily by SEC-HPLC. Monomers, dimers and soluble aggregates can be detected by this technique. The mobile phase used was 0.08 M TFA in 60/40% CH 3 CN / H 2 O, adjusted to pH 2.0, isocratic, at a flow rate of 1.5 ml / min. Signals were detected at 220 nm. A Bio-Rad Bio-Sil® SEC 250 column, particle size 5 µm, 300 x 7.8 ID, equipped with a safety column (Bio-Rad Bio-Sil® SEC 250 Guard, particle size 5 µm, 80 x 7.8) was used ID). The injection volume was 10 µl. Standard calibration curves were 0, 0.1, 0.25, 0.5, 0.75 and 1.0 mg / ml bST in the mobile phase.

The results are shown in Figure 3. As can be seen, bST was released for 14 days. No detectable levels of dimers or soluble aggregates were evident in the release medium. The minimum initial release was 12% on each of the first two days, after which the release rate was moderated.

Example 6: Short-term insulin release

Preparation of the preparation: A degradable macromer (3.4KL5) was used. The protein used was Zn-insulin (purchased from Sigma). Protein was added based on dry weight of 47%. Three samples were prepared.

Samples were prepared as described in Example 4. Samples were analyzed by SEC-HPLC for detection of monomers, dimers and soluble aggregates using the conditions described in Example 5.

The results are shown in Figure 4. Insulin was released for 24 hours; no dimers or soluble aggregates were detected. Complete release (100%) was achieved within 24 hours.

Example 7: Release of drug from a mixture of insoluble and soluble macromer

The formulation was manufactured as described above. Macromers containing a mixture of soluble macromer (3.4KL5) and insoluble macromer (3.4KC6) were used in a 50:50 ratio. The protein used was ZnbST (Protiva / Monsato); 25% was added based on dry weight. Six samples were prepared. Samples were analyzed by SEC-HPLC as described above. The samples were tested for the presence of monomers, dimers and soluble aggregates.

The results are shown in Figure 5. ZnbST release was observed for 20 days; very low concentrations (less than 2%) of dimers or soluble aggregates were detected. Furthermore, no sudden initial drug release was observed.

Example 8: Release of the drug from a mixture of insoluble and soluble macromers

The formulation was manufactured as described above. A 75:25 mixture of soluble macromers (3.4KL5) and insoluble macromers (3.4KC6) was used. The protein used was ZnbST (Protiva / Monsato); 25% was added based on dry weight. Six samples were prepared. Samples were analyzed by SEC-HPLC as described above. The samples were tested for the presence of monomers, dimers and soluble aggregates as described above.

The results are shown in Figures 5 and 6. Long-term ZnbST release was observed for 17 days; within 13 days of release, 90% of the incorporated ZnbST was released. Very few dimers or aggregates have been released.

Example 9: Controlled release of bovine somatotropin in pituitary-depleted rats

Controlled delivery of active bovine somatotropin (20,000 dalton molecular weight) was verified in a pituitary-depleted rat model. Pituitary-removed rats were purchased from Taconic Labs (Germantown, NY). Rats were weighed every morning. Rats were kept for 7 days prior to the start of the study to confirm that there was no growth. On the first day of the study, rats weighed 118 ± 1.5 grams (mean ± standard deviation, n = 18). Rats were divided into three groups with balanced average weights. Group 1 was not treated and served as a negative control. Group 2 received a bST implant in a hydrogel prepared from a 3: 1 mixture of 3.4KL5 and PEGDA (each formulation contained 0.9 to 1.1 mg bST). Group 3 rats were injected with 100 µg bST subcutaneously each day for the duration of the study.

The results are shown in Figure 8. The untreated group did not grow during the study and weighed an average of 119 ± 2.9 grams after 11 days. Group 3 rats, which received 100 pg bST daily for the duration of the study, showed continued growth and weighed 151 ± 4 grams after 11 days of treatment. Group 2 rats grew at a similar rate to group 3 rats and weighed 145 ± 3.7 grams after 11 days (p = 0.32 compared to group 3, t-test).

Example 10: Release of bST

A macromer blend containing approximately 30 weight percent bST was prepared using the methods described above. The macromer / protein mixture was placed in a glass cylinder having an internal diameter of either 1.12 mm or 0.61 mm. The system was exposed to light for 20 s, removed from the glass cylinder, placed on aluminum foil and exposed to light for an additional 3.5 min. The resulting hydrogel cylinder was placed in 1 ml release medium (PBS, pH 7.4) and the released bST was detected by HPLC. Initial data suggest that the release from the larger diameter cylinder closely follows the release from the smaller diameter cylinder. Further, the bST release characteristics indicate degradation / swelling of the controlled system. The system showed the following fractionated release Μ / Μ »for short periods of time as a power function of time t: M / Mn = k't ⁿ , where k'is constant characteristic of the system and ⁿ is the exponent characteristic of the mode of transport. For n = 0.5, drug release follows the diffusion mechanism of Fickian. For n > 0.5, behavior other than Fickian type was observed.

The data presented in Figure 9 were analyzed for an erosion / diffusion release mechanism. The large cylinder had a value of M / M ~ = 1E-06t ² and a smaller cylinder had a value of M / IVL = 3E-05t ² . Therefore, when n = 2, behavior other than Fickian type was observed.

In a different diffusion-based analysis, the flow from the cylinder was analyzed using the following Fickian equations:

J = D * A * AC / kde, where J is the flow; D is a diffusion constant; A is a surface area; C is the concentration in the cylinder and S is the distance from the center. In this analysis, the flow should vary dramatically depending on whether the release takes place from a large or small diameter cylinder. Theoretical analysis predicts by Fickian diffusion that if a smaller diameter cylinder would release 20%, a larger diameter cylinder would release 7% of the embedded drug. However, it was observed that when the smaller diameter cylinder relaxed, the larger diameter cylinder released 16%. Therefore, non-Fickian type behavior was observed.

In these hydrogel systems, the initial release phase comprises water uptake (swelling); as a result, a homogeneous course of drug concentration within the matrix becomes S-shaped. There is a high drug concentration in the center of the cylinder and no or very little drug is available on the perimeter of the drug. Such a cylindrical system runs the release kinetics independently of the cylinder radius. A detailed description of this phenomenon can be found in Ping I. Lee, &quot; Diffusion Controlled Matrix Systems &quot; in Treatise on Controlled Drug Delivery, edited by Kydonieus, A., pp. 155-197 (1992).

Example 11: Controlled Erythropoietin Release in Rats

Controlled delivery of active human erythropoietin (EPO) was confirmed in male Sprague-Dawley rats purchased from Taconic Labs (Germantown, NY). According to the procedure described in Example 14, hydrogel formulations containing 3000 units per formulation were formed. The formulations were formed in the absence of vinylpyrrolidone and other polymerizable monovinyl monomers. One of these preparations was implanted in each of the three rats. Three other rats received a daily subcutaneous injection of EPO (1000 units) for three days. The control group of three rats was not treated.

On the fifth day after implant implantation or the start of subcutaneous injections, a venous blood sample was obtained from each rat and stored in EDTA. Reticulocyte fractions (immature red blood cell cells) were determined after acridine orange staining by automatic flow cytometry.

The results are shown in Figure 10. As shown there, the rats in the control group had about 2.5% reticulocytes. Implant rats had about 12% reticulocytes and rats receiving injections had about 19% reticulocytes after five days.

Example 12: Controlled insulin release in diabetic rats

Sprague-Dawley rats were purchased from Taconic Labs (Germantown, NY). Diabetes was induced by streptozotocin (65 mg / kg, intravenous) and confirmed after 48 hours by an increase in the blood glucose balance (> 3000 mg / l). After anesthesia of the rat with pentobarbital (35 mg / kg), a catheter was placed in the throat vein. After a baseline blood sample was taken to determine the blood glucose concentration, a 9 hydrogel formulation containing 1 unit of insulin was implanted subcutaneously in the rat. The formulations were formed in the absence of vinylpyrrolidone and other polymerizable monovinyl monomers. Blood samples were taken for 15, 30, 60, 120 and 180 min. after implant preparation and were used to determine blood glucose levels.

The results are shown in Figure 11. As shown there, blood glucose levels have decreased. This indicates that the formulations are capable of releasing insulin in its active form.

To test the pulmonary delivery system, the throat was opened through the mid section and the trachea was opened through the cross section. A notch was formed in the trachea and a small polyethylene tube was inserted into the lungs. A small volume of insulin-containing hydrogel microspheres (total dose was 3 units of insulin) was instilled into the lungs and the tube was removed. Blood samples were collected and analyzed as described above for the subcutaneous formulation.

The results are shown in Figure 12. Glucose levels decreased significantly within 30 min. and remained low (below 1500 mg / l) for at least 180 min.

Example 13: Controlled release of human growth hormone in pituitary-depleted rats

Controlled delivery of active human growth hormone (hDH, 20,000 dalton molecular weight) was verified in a pituitary-depleted rat model. Pituitary-removed rats purchased from Taconic Labs (Germantown, NY) were weighed each morning. Rats were kept for 7 days prior to the start of the study to confirm that there was no growth. Rats were divided into 3 groups with balanced average weights. Group 1 was not treated and served as a negative control. Group 2 received a hGH implant in a hydrogel prepared from a 3: 1 mixture of 3.4KL5 and 3.4KC6 (each formulation contained approximately 1 mg hGH). Group 3 rats received 100 µg hGH subcutaneously each day for the duration of the study.

Initial results indicate that the previous results obtained with bST were repeated using hGH. The untreated group did not grow during the study. Group 3 rats, which received 100 pg hGH daily for the duration of the study, showed continued growth. Group 2 rats grew at a similar rate to group 3 rats.

Example 14: Release of EPO from a macromer

9,999,999,999

9

999 9999

..............

In a sterile 20 ml vial was added: 0.0330 g TEOA (undiluted), 1.0076 g 3.4KL5, 0.0598 g 1000 ppm eosin (in PBS, pH 7.0) and 2.32 g EPO solution (10,000 units / ml). No vinylpyrrolidone or other polymerizable monovinyl monomer was added. The resulting mixture was mixed and polymerized by light (ILC Technology, Inc., Fiber optic xenon light source).

The in vitro release rate was determined as the average release from three disks containing an average of 2500 units per disk. The release was performed in 4 ml PBS (pH 7.4) at 39 ° C. The release medium was changed daily. Analysis was performed by chromatography. (HPLC: model 2690 from Waters, column: SEC 250 from BioRad, mobile phase: 0.8 M TFA in 60% acetonitrile @ 1.5 mL / min, detector wavelength: 220 nm).

The results are shown in Figure 13. As indicated there, EPO was released for at least 120 h. After 120 hours, more than 500 EPO units were released.

Example 15: Release of insulin from macromer particles

In a sterile 20 ml vial was added: 0.2559 g 200 mM TEOA (in PBS buffer, pH 7.0), 0.2548 g 3.4KL5, 0.0206 g 1000 ppm eosin (in PBS, pH 7.0) and 0.0615 g of insulin (Sigma). No vinylpyrrolidone or other polymerizable monovinyl monomer was added. The resulting mixture was stirred and placed in 10 ml glass tubes. The tubes were exposed to a xenon light source (ILC Technology, Inc., A fiber optic xenon light source) for 10 seconds. The half-treated hydrogel was extruded from the glass tube and polymerized for 3.5 minutes. The treated hydrogel rods were placed in 15 ml of heptane and milled using a homogenizer (Silverson L4RT-A) for 30 s and 5000 rpm, followed by 30 s at 3000 rpm. The heptane was decanted and the powder dried under nitrogen. The resulting particles had a size distribution of from 2 µm to 500 µm.

Particles (16 mg) were placed in a porous (0.8 mm) release measuring bag (described in Example 5). In vitro release was calculated as the mean release from two measurements. The release bag was placed in 2 ml PBS (pH 7.4) at 39 ° C. The release medium was changed every 15 min during the first 2 h. and then every 30 min. Analysis was performed by chromatography. (HPLC: model 2690 from Waters, column: SEC 250 from BioRad, · ft 4 4 · • ·

Mobile phase: 0.8 M TFA in 60% acetonitrile @ 1.5 ml / min, detector wavelength: 220 nm).

The results are shown in Figure 14. As indicated there, insulin was released for 90 minutes. More than 100 µg of insulin was released after 90 minutes.

Example 16: Release of Luteinizing Hormone Releasing Hormone (LHRH)

In a 20 ml vial was added: 0.2559 g 200 mM TEOA (in PBS buffer, pH 7.0),

0.2548 g 1KC3, 0.0206 g 1000 ppm eosin (in PBS, pH 7.0) and 0.0615 g LHRH (Sigma). No vinylpyrrolidone or other polymerizable monovinyl monomer was added. The resulting mixture is placed between two glass plates and polymerized by a xenon light source (ILC Technology, Inc., A fiber optic xenon light source) for 2 m from each side. The resulting hydrogel sheet was ground at temperatures well below freezing to form an injectable powder.

Example 17: Pulmonary preparations containing human growth hormone (hGH)

In a 20 ml vial was added: 0.2559 g 200 mM TEOA (in PBS buffer, pH 7.0),

0.2548 g 3.4KL5, 0.0206 g 1000 ppm eosin (in PBS, pH 7.0) and 0.0615 g hGH (Genentech hGH preparation injected, Millipore Centricon ™ purified). No vinylpyrrolidone or other polymerizable monovinyl monomer was added. The resulting mixture was stirred and placed in 10 ml glass tubes. The tubes were exposed to a xenon light source (ILC Technology, Inc., A fiber optic xenon light source) for 10 seconds. The half-treated hydrogel was extruded from the glass tube and polymerized for 3.5 minutes. The treated hydrogel rods were placed in 15 ml of heptane and milled using a homogenizer (Silverson L4RT-A) for 30 s and 5000 rpm, followed by 30 s at 3000 rpm. The heptane was decanted and the powder dried under nitrogen. The powder is used for pulmonary, oral or subcutaneous sustained delivery of hGH.

Example 18: Release of GLP-1

GLP-1 (glucagon-like peptide-1) is a peptide drug that has been shown to be promising for the treatment of type-II diabetes. In a 20 ml vial were added: 0.2559 g 200 mM TEOA (in PBS buffer, pH 7.0), 0.2548 g 1KC3, 0.0206 g 1000 ppm eosin (in PBS, pH 7.0) and 0, 0615g GLP-1. The resulting mixture is placed between two glass plates and fefefe fefefe fefefe • fe · fefefe fefefe • fefe fefe fefe fefe fefe fefe * fefe fefe fefe fefe fefe fefe fefe light polymerized by xenon light source Technology, Inc. (xenon fiber optic light source) for 2 m from each side. The resulting hydrogel sheet was ground at temperatures well below freezing to form an injectable powder.

Example 19: Oral preparations for protein release

Using the procedure of Example 15, one of the following substances, insulin, human growth hormone, human alpha interferon, or erythropoietin, was incorporated into the macromer particles. Very small microparticles, preferably having an average size less than or equal to 500 nanometers, are produced by milling at temperatures well below the freezing point or milling mentioned in Example 15. Such nanoparticles are then administered to the rats surgically into the gastrointestinal tract, by catheter infusion into the upper part of the gastrointestinal tract. The dosage of such nanoparticles is based on the assumption that about 0.5% of the drug in the nanoparticles will be detectable in the blood of these rats, for example by RIA, taking into account the specific pharmacology of each drug considered.

For insulin, blood samples were taken at t = -15, 0, 30, 60, 90, 120 and 180 min. and determining insulin content by RIA and blood glucose level by glucometer (diabetic rats are used when insulin is administered).

For other drugs, normal rats are used and blood levels of drugs are measured at the same time points using RIA and ELISA techniques.

In addition to the above procedures, microspheres containing the aforementioned drugs can be modified to increase their absorption in the small intestine, colon and other suitable areas of the gastrointestinal tract. Such modifications may include precipitation of a lipid bilayer around the microcapsules so as to mimic fat particles from the digested food, attaching molecules such as ferritin or applying a charged layer to the outside of the microparticles.

Other embodiments

It will be apparent from the foregoing description that variations and modifications may be made to the invention described herein to be adapted to various uses and conditions. Such embodiments also fall within the scope of the following claims.

All publications and patents mentioned in this Regulation are incorporated herein to the same extent as if each individual publication or patent were specifically and individually identified and incorporated by reference.

Claims (72)

PATENT CLAIMS A biologically active substance for use in therapy comprising: (a) combining said biologically active substance with a macromer; b) forming a mixture from the combination formed in step a); c) polymerizing said mixture and forming formulations; and d) administering said compositions or a portion thereof to a mammal, said composition comprising at least 5 wt. % of biologically active substance. 2. A biologically active substance for use in therapy comprising: (a) combining said biologically active substance with a macromer; b) forming a mixture from the combination formed in step a); c) polymerizing said mixture and forming formulations; and d) administering said compositions or a portion thereof to a mammal, wherein the time during which 10% of the released active agent is released is greater than 1/10 t ₅ o. and wherein the biologically active agent is a protein, said composition comprising at least 5 wt. % of the protein, wherein the macromer is soluble or wherein said polymerization occurs in the absence of a water-soluble polymerizable vinylpyrrolidine monomer. A biologically active agent for use in therapy, wherein the time during which 10% of the releasable active agent is released is greater than 1/10 t ₅₀ . A biologically active agent for use in therapy, wherein the composition comprises at least 10 weight percent of the active agent. A biologically active agent for use in therapy, wherein the composition comprises at least 25 weight percent of the active agent. A biologically active agent for use in therapy, wherein the composition comprises at least 40 weight percent of the active agent. A biologically active agent for use in therapy wherein the macromer comprises: (a) a water-soluble region which forms the central core; b) at least two degradable regions attached to said core; and c) at least two polymerizable end groups wherein said polymerizable end groups are attached to said degradable regions. A biologically active agent for use in therapy wherein the water-soluble region comprises a polymer selected from the group: polyethylene glycol, polyethylene oxide, polyvinyl alcohol, polyvinylpyrrolidone, polyethyloxazoline, polyethylene oxide / polypropylene oxide block copolymers, polysaccharides, sugars, proteins, and combinations thereof. A biologically active agent for use in therapy wherein the degradable region comprises a polymer selected from the group: poly-α-hydroxy acids, polylactones, polyamino acids, polyanhydrins, polyorthoesters, polyorthocarbonates and polyphosphoesters. A biologically active agent for use in therapy wherein the degradable region comprises polytrimethylene carbonate. A biologically active agent for use in therapy wherein the degradable region comprises polycaprolactone. A biologically active agent for use in therapy wherein the poly-α-hydroxy acid is selected from the group: polyglycolic acid, poly-DL-lactic acid and poly-L-lactic acid. A biologically active agent for use in therapy wherein the polylactone is selected from the group: poly-ε-caprolactone, poly-δ-valerolactone and poly-γ-butyrolactone. A biologically active agent for use in therapy wherein the polymerizable end groups contain a double bond between two carbon atoms capable of polymerizing the macromer. · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · A biologically active agent for use in therapy wherein the core comprises polyethylene glycol; said degradable regions comprise a biodegradable poly-hydroxy acid and said end groups comprise an acrylate oligomer or monomer. A biologically active agent for use in therapy wherein step d) comprises administering said compositions to the lungs of said mammal. A biologically active agent for use in therapy wherein step d) comprises intravenous administration of said compositions. A biologically active agent for use in therapy wherein step d) comprises subcutaneous administration of said compositions. A biologically active agent for use in therapy wherein step d) comprises intramuscular administration of said compositions. A biologically active agent for use in therapy wherein step d) comprises oral administration of said compositions. A biologically active agent for use in therapy wherein step d) comprises nasal administration of said compositions. A biologically active agent for use in treatment wherein the mammal is a human. A biologically active agent for use in therapy wherein the biologically active agent is a protein. A composition made by the method of claim 1. A composition made by the method of claim 2. A composition made by the method of claim 3. A biologically active agent for use in therapy comprising: (a) combining said biologically active substance with a macromer; b) forming a mixture from the combination formed in step a); c) polymerizing said mixture and forming formulations; and d) administering said compositions or a portion thereof to a mammal, said compositions releasing at least 80% of said biologically active agent over a period of time that is 2.5 times longer than t ₅₀ . A biologically active agent for use in therapy comprising: (a) combining said biologically active substance with a macromer; b) forming a mixture from the combination formed in a); c) polymerizing said mixture and forming formulations; and d) administering said compositions or a portion thereof to a mammal, said compositions releasing a therapeutic dose of said biologically active agent over a period of time that is 2.5 times longer than the t 50. A composition for delivering a biologically active agent, said composition comprising hydrogel-containing particles and a biologically active agent, wherein the release kinetics of said particles are independent of particle size, said particles having a mean diameter of from 50 nm to 1 mm. 31. A process for the manufacture of controlled release preparations of biologically active substances, comprising the steps of: a) combining said biologically active agent with a biodegradable macromer, said macromer comprising at least one water-soluble region, at least one degradable region that is hydrolyzable under in vivo conditions and polymerizable end groups capable of forming additional covalent bonds in the presence of an initiator resulting in polymerization of the macromer, wherein said polymerizable end groups are separated by at least one degradable region; b) polymerizing said macromer in the absence of light to form hydrogels and incorporate said biologically active agent into said hydrogels; and c) forming preparations of said hydrogels capable of controlled release of said biologically active substance. 32. The method of claim 31, wherein said initiator is a radical initiator. 33. The method of claim 31, wherein said initiator is an ionic initiator. 34. A method for producing a polymerized hydrogel comprising the steps of: (a) a combination of a hydrophobic, water-insoluble macromer, initiator and water; b) allowing said macromer to swell; c) stirring said macromer to form a homogeneous mixture; and d) polymerizing said macromer and forming a hydrogel. 35. The method of claim 34, wherein said method comprises adding a biologically active agent to said mixture prior to step d). 36. A process for producing a polymerized hydrogel comprising the steps of: (a) a combination of a hydrophilic macromer and a hydrophobic, water-insoluble macromer; b) heating and stirring the combination formed in step a) to form a homogeneous mixture; c) cooling said mixture to room temperature; d) adding water and an initiator to said mixture and allowing said mixture to swell; and e) polymerizing said macromer, and forming a hydrogel. 37. The method of claim 36, wherein said method comprises adding a biologically active agent to said mixture prior to step e). a) combining said protein with a polymerizable, hydrophilic polymer; b) forming a mixture from the combination formed in step a); c) polymerizing said mixture and forming formulations; and • » d) administering said compositions or a portion thereof to a mammal, wherein said protein remains intact and wherein at least 70% of said protein is released from said compositions. A biologically active agent for use in therapy comprising: a) combining said biologically active agent with a biodegradable, polymerizable macromer in aqueous solution and in the presence of a free radical initiator; b) dispersing said solution and forming fine droplets comprising said macromer and said biologically active agent; c) polymerizing said macromers contained in the droplets thereby forming hydrogel particles having a biologically active agent incorporated therein, said particles being capable of controlled release of the biologically active agent; and d) administering said compositions or a portion thereof to a mammal, said composition comprising at least 5 wt. or wherein the time during which 10% of the active substance released is greater than 1/10 tgo. 39. The method of claim 38 wherein said solution is dispersed by drying the spray liquid or by a water-in-oil emulsion process. 40. The method of claim 38, wherein at least 80% of said particles have a particle size of less than 5 µm. 41. A composition comprising a biologically active agent, wherein said biologically active agent is enclosed within a biodegradable, polymerizable macromer, wherein said macromer comprises at least one water soluble region, at least one degradable region that is hydrolyzable under in vivo conditions, and polymerizable end groups having the ability to form additional covalent bonds resulting in the polymerization of macromers, wherein said polymerizable end groups are separated by at least one degradable region and wherein said composition comprises at least 5 weight percent of said biologically active agent. 42. The composition of claim 41 comprising at least 10 weight percent of said biologically active agent. 43. The composition of claim 41 comprising at least 20 weight percent of said biologically active agent. 44. An insoluble macromer comprising at least one water-soluble region, at least one degradable region that is hydrolyzable under in vivo conditions, and polymerizable end groups that have the ability to form additional covalent bonds that result in polymerization of the macromer, wherein: said polymerizable end groups being separated by at least one degradable region. 45. The macromer of claim 44, wherein said degradable region comprises a mixture of at least two different polymers. 46. The macromer of claim 44, wherein said degradable region comprises a copolymer of at least two different monomers. 47. The macromer of claim 44, wherein said water-soluble region comprises at least two arms. 48. A macromer according to claim 44, wherein said water-soluble region consists essentially of polyethylene glycol having a molecular weight of 400 to 8000 daltons. 49. A sustained-delivery protein composition comprising an insoluble macromer comprising at least one water-soluble region, at least one degradable region that is hydrolyzable under in vivo conditions, and a polymerizable end group having the ability to form additional covalent bonds, resulting in polymerization of the macromer, wherein said polymerizable end groups are separated by at least one degradable region. 50. A macromer comprising at least one water-soluble region, at least one degradable region that is hydrolyzable under in vivo conditions, and polymerizable end groups that have the ability to form additional covalent bonds that result in polymerization of the macromer, wherein said macromer comprises: the polymerizable end groups are separated by at least one degradable region, wherein said degradable region consists essentially of polytrimethylene carbonate. 51. A composition for subcutaneous administration of LHRH comprising a 1000 dalton polyethylene glycol core and a degradable region consisting of polycaprolactone, wherein said composition is capable of delivering a therapeutic dose of LHRH for a period of more than 30 days. 52. A composition comprising a glucagon-like peptide-1 and a macromer, wherein said macromer comprises at least one water-soluble region, at least one degradable region that is hydrolyzable under in vivo conditions, and a polymerizable end group having the ability forming additional covalent bonds that result in polymerization of the macromer, wherein said polymerizable end groups are separated by at least one degradable region. 53. A sustained release hydrogel composition comprising particles having a density of less than 0.4 g / cm ³ , wherein at least 50% of said particles have an average diameter of less than 5 µm, and wherein said the composition is formulated for pulmonary administration. 54. A sustained release composition of a biologically active agent comprising particles having a density of greater than 0.4 g / cm ³ . 55. The composition of claim 54, wherein said composition is formulated for pulmonary administration. The biologically active agent for use in the treatment of claims 1, 2, 3, 24, 28, 29, or 38 which is selected from the group consisting of a therapeutic agent, a diagnostic agent, a prophylactic agent, a drug and a protein. The biologically active agent for use in the treatment of claims 59, wherein the therapeutic agent is selected from the group consisting of calcitonin, granulocyte macrophage colony stimulating factor, ciliary neurotrophic factor, parathyroid hormone and transmembrane cystic fibrosis regulatory gene, parathyroid hormone-like peptide, somatostatin, testosterone , progesterone, estradiol, nicotine, fentanyl, norethisterone, clonidine, scopolamine, salicylate, saimeterol, formeterol, albeterol and valium, cancer chemotherapeutic agents, cytokines, lymphokines, DNA and vaccines, attenuated influenza virus, cDNA, expression vectors, molecules having positive polarity , ribozymes, polysaccharides, heparin, tissue plasminogen activator (tPa), superoxide dismutase, luteinizing hormone releasing hormone catalase antagonists, platelet factor IL-11, IL-4 receptor, "enbrel", IL-2 receptor antagonists, TFN receptor fusion proteins, growth and growth megakaryocyte development factor, stem gene, anti-HER-2 and anti VEGF humanized monoclonal antibodies, anti-Tac antibodies, GLP-1 amylin and GLP-1 amylin analogues, atrial natriuretic factor, atrial natriuretic peptide, human chorionic β-gonadotropin, essential fibroblast growth factor, bovine growth hormone, bone morphogenetic protein, B-cell stimulation factor-1, B-cell stimulation factor, bovine somatotropin, anti-cancer factor, cartilage inducing factor, corticotropin releasing factor, colony stimulating factor, differentiation factor- 1, lining cell growth factor, erythrocyte differentiation factor, elongation factor 1-alpha, epidermal growth factor, erythropoietin, fibroblast growth factor, follicle stimulating hormone, granulocyte colony stimulating factor, glial fibril acid acid protein, growth hormone releasing factor, human alpha-1 antitrypsin, human atrial natriuretic fa which, human chorionic gonadotropin, human growth hormone, human leukemia inhibiting factor, hemopoietin-1, hepatocyte growth factor, human transforming growth factor, human thyroid stimulating hormone, interferon, immunoglobulin A, immunoglobulin D, immunoglobulin E, growth factor-1-like insulin, insulin-like growth factor-11, immunoglobulin G, immunoglobulin M, interleukin-1, interleukin-2, interleukin-3, interleukin-4, interleukin-5, interleukin-6, renal plasminogen activator, cell adhesion molecule , luteinizing hormone, leukemia inhibiting factor, monoclonal antibody, macrophage activating factor, macrophage cytotoxic factor, macrophage colony stimulating factor, megakaryocyte colony stimulating factor, tumor necrosis factor A, macrophage inhibiting factor, Mullerian inhibiting factor, megakaryocyte stimulating factor, , neutro flush chemotactic factor, nerve growth factor, novel plasminogen activator, non-steroidal anti-inflammatory drug, osteogenic extract factor, anti-tumor lymphokine, prostate-specific antigen, plasminogen activator inhibitor, platelet-derived growth factor, platelet-derived wound healing agent, human plasma protein inducing interleukin, tumor vascular factor, tissue control factor, T-cell growth factor, T-cell modulating peptide, transforming growth factor, tumor growth inhibitor, tumor inhibiting factor, tissue metalloproteinase inhibitor, tissue necrotic factor, tissue plasminogen activator, vascular endothelial growth factor; and vasoactive intestinal peptide. A biologically active agent for use in therapy wherein the biologically active agent is bovine somatotropin. 59. A biologically active agent for use in therapy wherein the biologically active agent is erythropoietin. 60. A biologically active agent for use in therapy wherein the biologically active agent is human growth hormone. The biologically active agent for use in the treatment of claims 37 or 56, wherein the protein is selected from the group consisting of insulin, hormones, growth hormones, human growth hormone and bovine growth hormone, factor VIII, factor IX, factor VIIa, anti-inflammatory agents, interleukins. , interleukin-4, NSAIDs, corticosteroids, enzymes, DNase and proteases, cytokines, interferons, interferon alpha, interferon beta, poetins, colony stimulating factors, growth factors, ceredase, gibberellins, auxins, vitamins, vascular endothelial growth factor, cell growth factor lining, an essential factor for fibroblast growth and platelet-derived growth factor. The biologically active agent for use in therapy according to claim 61, wherein the protein is insulin. The biologically active agent for use in treatment according to claim 56, wherein the medicament is selected from the group consisting of pentamidine isethioute, albuterol sulfate, β-agonist, metaproterenol sulfate, beclomethasone dipropionate, triamcinolone acetamide, budesonide acetonide, ipratropium bromide, flunisolide, sodium cromolyn, ergotamine tartrate and protein or peptide drugs such as TFN antagonists and interleukin antagonists. The biologically active agent for use in treatment according to claim 56, wherein the diagnostic agent is selected from the group consisting of a gas, the gadolinium chelate, iron, magnesium, manganese, copper and chromium chelates and iodine-based materials. 65. The composition of claims 29, 41, 42, 43, 51 or 52 wherein the biologically active agent is selected from the group consisting of a therapeutic agent, a diagnostic agent, a prophylactic agent, a drug, and a protein. 66. The composition of claim 65, wherein the therapeutic agent is selected from the group consisting of calcitonin, granulocyte macrophage colony stimulating factor, ciliary neurotrophic factor, parathyroid hormone and a transmembrane cystic fibrosis regulatory gene, parathyroid hormone-like peptide, somatostatin, testosterone. , progesterone, estradiol, nicotine, fentanyl, norethisterone, clonidine, scopolamine, salicylate, salmeterol, formeterol, albeterol and valium, cancer chemotherapeutic agents, cytokines, lymphokines, DNA and vaccines, attenuated influenza virus, cDNA, expression vectors, molecules having positive polarity , ribozymes, polysaccharides, heparin, tissue plasminogen activator (tPa), superoxide dismutase, luteinizing hormone releasing hormone catalase antagonists, platelet factor IL-11, IL-4 receptor, "enbrel", IL-2 receptor antagonists, TFN receptor fusion proteins, growth and development fa megakaryocytes, stem gene, anti-HER-2 and anti VEGF humanized monoclonal antibodies, anti-Tac antibodies, GLP-1 amylin and GLP-1 amylin analogues, atrial natriuretic factor, atrial natriuretic peptide, human chorionic β-gonadotropin, basic growth fibroblast factor, bovine growth hormone, bone morphogenetic protein, B-cell stimulating factor, B-cell stimulating factor-2, bovine somatotropin, anti-cancer factor, cartilage inducing factor, corticotropin releasing factor, colony stimulating factor, differentiation factor-1 , lining cell growth factor, erythrocyte differentiation factor, elongation factor 1-alpha, epidermal growth factor, erythropoietin, fibroblast growth factor, follicle stimulating hormone, factor • φ · · · · ♦ ·· φ · · φ · * φφφ φφφ ·· Φ ·· ·· · φ · stimulating granulocyte colonies, glial fibrilic acid protein, releasing factor growth hormone, human alpha-1 antitrypsin, human atrial natriuretic factor, human chorionic gonadotropin, human growth hormone, human leukemia inhibiting factor, hemopoetin-1, hepatocyte growth factor, human transforming growth factor, human thyroid stimulating hormone, interferon, , immunoglobulin D, immunoglobulin E, insulin-like growth factor-1, insulin-like growth factor-11, immunoglobulin G, immunoglobulin M, interleukin-1, interleukin-2, interleukin-3, interleukin-4, interleukin-5, interleukin -6, renal plasminogen activator, cell adhesion lectin molecule, luteinizing hormone, leukemia inhibiting factor, monoclonal antibody, macrophage activating factor, macrophage cytotoxic factor, macrophage colony stimulating factor, megakaryocyte colony stimulating factor, tumor necrosis factor A, macrophage inhibiting factor , Mullerian inhibition substance, megakaryocyte stimulating factor, melanocyte stimulating factor, neutrophil chemotactic factor, nerve growth factor, novel plasminogen activator, non-steroidal anti-inflammatory drug, osteogenic extract factor, anti-tumor lymphokine, prostate-specific antigen, plasminogen activator inhibitor, growth factor derived from, for the treatment of platelet-derived injuries, human plasma protein inducing interleukin, vascular formation factor in tumors, tissue control factor, T-cell growth factor, T-cell modulating peptide, transforming growth factor, tumor growth inhibitor, tumor inhibiting factor, tissue inhibitor metalloproteinases, tissue necrotic factor, tissue plasminogen activator, vascular endothelial growth factor, and vasoactive intestinal peptide. 67. The composition of claim 66, wherein the therapeutic agent is bovine somatotropin. 68. The composition of claim 66, wherein the therapeutic agent is erythropoietin. 69. The composition of claim 66, wherein the therapeutic agent is human growth hormone. The composition of claims 49 or 65, wherein the protein is selected from the group consisting of insulin, hormones, growth hormones, human growth hormone and bovine growth hormone, factor VIII, factor IX, factor VIIa, anti-inflammatory agents, interleukins, interleukin-4, NSAIDs. , corticosteroids, enzymes, DNase and proteases, cytokines, interferons, interferon alpha, interferon beta, poetins, colony stimulating factors, growth factors, ceredase, gibberellins, auxins, vitamins, vascular endothelial growth factor, endothelial cell growth factor, essential factor for growth fibroblasts and platelet-derived growth factor. The composition of claim 70, wherein the protein is insulin. The composition of claim 65, wherein the medicament is selected from the group consisting of pentamidine isethioute, albuterol sulfate, β-agonist, metaproterenol sulfate, beclomethasone dipropionate, triamcinolone acetamide, budesonide acetonide, ipratropium bromide, flunisolide, cromolyn sodium, ergotamine tartrate, and protein ergotamine. drugs such as TFN antagonists and interleukin antagonists. The composition of claim 65, wherein the diagnostic agent is selected from the group consisting of a gas, the gadolinium chelate, iron, magnesium, manganese, copper and chromium chelates, and iodine-based materials.