Title:  Ionically formulated biomolecule microcarriers

United States Patent:  6,645,525

Issued:  November 11, 2003

Inventors:  Woiszwillo; James E. (Milford, MA)

Assignee:  Sedum Laboratories, Inc. (Mansfield, MA)

Appl. No.:  018991

Filed:  May 6, 2002

PCT Filed:  June 23, 2000

PCT NO:  PCT/US00/17223

PCT PUB.NO.:  WO01/01964

PCT PUB. Date:  January 11, 2001

Abstract

Compositions for sustained delivery of a biomolecule including an anionic polymer and a cationic polymer which ironically interact with each other and, optionally, with the biomolecule. Methods for making the compositions, including the step of combining the negatively charged polymer with the positively charged polymer to form an ionic complex are also provided. The biomolecule may be complexed with one of the polymers before it is complexed with the oppositely charged polymer. The complex is exposed to conditions that cause the formation of precipitated microcarriers, such as a change in pH or the addition of a complexing molecule. The compositions are preferably formulated into microcarriers.

SUMMARY OF THE INVENTION

Compositions for sustained delivery of biomolecules are described herein. The compositions include an anionic polymer (polyanion) and a cationic polymer (polycation) which ionically interact with each other and, optionally, with the biomolecule to form a polymer matrix or complex. Also provided are methods for making the compositions, including the step of combining the negatively charged polymer with the positively charged polymer to form anionic complex. The biomolecule may be complexed with either one of the polymers, depending on the characteristics of the biomolecule, such as the charge of the biomolecule. Then the complex is reacted with the oppositely charged polymer. The complex is exposed to conditions, such as a change in pH or the addition of a complexing molecule, that cause the formation of precipitated microparticles, also referred to herein as microcarriers. The compositions are preferably formulated into microcarriers. The preferred polyanions and polycations are water soluble polymers, available commercially at high purity, that are already known and on the GRAS (generally regarded as safe) list. Alternatively, the polymers are high molecular positively or negatively charged polymers synthesized using polymer chemistry synthesis methods known to those skilled in the art.

In a preferred embodiment, the cationic polymer is polyethyleneimine (PEI), polychitosan, or a cationic polymethacrylate, and the anionic polymer is dextran sulfate, heparin, alginic acid or an anionic polymers are available in a range of molecular weights, typically in the range of 20,000 to 500,000 kD.

Most preferably, insulin, a positively charged protein, is first complexed with dextran sulfate. The cationic polymer, PEI or DEAE dextran, is than added and complexed with the insulin/dextran sulfate complex. Formation of microcarriers is initiated by addition of zinc sulfate.

In another preferred embodiment, the cationic polymer is polyethyleneimine (PEI) and the anionic polymer is dextran sulfate or alginic acid. A negatively charged biomolecule, such as a nucleic acid, is first complexed with the PEI. The biomolecule/PEI complex is than complex with dextran sulfate. Formation of microcarriers is initiated by the addition of zinc sulfate.

Accordingly, it is an object of the invention to provide compositions for the delivery of biomolecules comprising microcarriers that release biomolecules at a sustained, constant rate of release.

It is another object of the present invention to provide compositions for delivery of biomolecules comprising microcarriers that provide stability to biomolecules during formulation and after administration.

It is another object of the present invention to provide microparticle compositions for the delivery of drugs in which the toxic effects of the drugs are minimized by being incorporated in a sustained-release microparticle formulation.

It is another object of the present invention to provide microparticle vaccines for the delivery of antigens in which an immunogenic effect is achieved in the absence of an adjuvant.

It is another object of the present invention to provide microparticle vaccines in which the polymers of the microparticles have an adjuvant effect.

These and other objects of the present invention will become apparent after reading the following detailed description of the disclosed embodiments and the appended claims.

DETAILED DESCRIPTION OF THE INVENTION

A microparticle composition, also referred to herein as a microcarrier composition, and method for forming microparticles, or microcarriers, for the delivery of biomolecules, such as proteins, peptides, polynucleic acids, and drugs, as well as larger biomolecules such as bacteria and viruses are provided. Preferably, the biomolecules in the microcarrier composition are in biologically active form or are immunogenic and are then released gradually in situ from the microcarrier when administered to a human or animal subject. The microcarriers are formulated using a mixture of water-soluble, positively and negatively charged polymers that interact with each other via ionic bonds. The biomolecule is optionally complexed with either the anionic polymer or the cationic polymer, depending upon the net charge of the biomolecule. The complexed biomolecule and polymer is then complexed with the oppositely charged polymer to form a combined complex in solution. Microcarriers are then formed by initiating coacervation, such as by changing the pH of the solution or adding a precipitating agent, such as zinc sulfate or certain amino acids.

MICROPARTICLE COMPOSITIONS

The microparticle compositions described herein contain a biomolecule, a cationic polymer and an anionic polymer. In a preferred embodiment, the biomolecule is complexed to either the cationic or anionic polymer. The complexed biomolecule/polymer is then complexed to the other polymer and microcarriers are formed. Preferably, the polymers are aqueous biodegradable polymers. Aqueous solutions are preferred over organic solutions because degradation of the biomolecule is avoided as is the use of chemical that may be toxic and therefore removed and recovered during the manufacturing process.

The preferred composition is a microparticle, microcarrier, or micromatrix, in which the biomolecule is homogeneously distributed throughout a polymer meshwork, thereby resulting in more uniform biomolecule release during in vivo degradation.

Biomolecules

A wide variety of biomolecules can be delivered using the compositions. The term biomolecule as used herein refers to bioactive, diagnostic, and prophylactic molecules. Biomolecules that can be used in the present invention include, but are not limited to, synthetic, recombinant or isolated peptides and proteins such as antibodies and antigens, receptor ligands, enzymes, and adhesion peptides; nucleotides and polynucleic acids such as DNA and antisense nucleic acid molecule; activated sugars and polysaccharides; bacteria; viruses; and chemical drugs such as antibiotics, antiinflammatories, and antifungal agents. Examples of proteins and peptides include insulin, luteinizing hormone release hormone (LHRH), somatostatin, calcitonin, vasopressin, epidermal growth factor (EGF), tissue plasminogen activator (TPA), human growth hormone, interleukins such as IL-2, interferon, erythropoietin (EPO) and others. Examples of other drugs that can be used in the compositions are amphotericin B (AMP-B), doxorubicin, and morphine sulfate.

Exemplary diagnostic agents include diagnostic enzymes and radiolabelled and fluorescent compounds.

Cationic Polymers

The cationic polymer of the microcarrier is a water soluble, positively charged polymer. Examples of cationic polymers that can be used in the invention include, but are not limited to, DEAE dextran (diethyleneaminoethyl dextran), polyethyleneimine (PEI), chitin, chitosan (D-acetylated chitin), and polyamino acids with a positive charge, such as polylysine. Peptoids, which are N-substituted polyglycines, achiral peptide variants in which side chains are sited on the amide nitrogen atom of each glycine monomer, have been shown to complex with plasmid DNA and can be used as the cationic polymer.

Anionic Polymers

The anionic polymer of the microcarrier is a water soluble, negatively charged polymer. Examples of anionic polymers that can be used in the present invention include, but are not limited to, dextran sulfate, heparin, and polyamino acids having a negative charge. Gel forming anionic polymers can be used, such as alginate and carageenan.

Precipitation Agent

The complexed polymers can be precipitated to form the microcarriers by changing the pH of the solution. Microcarriers can also be formed by adding a complex forming agent to the solution. For example, zinc sulfate and other multivalents salts will cause complexing of certain charged polymers. The size of the microcarriers is proportional to the concentration of the polymers and the amount of acid used to lower the pH or the amount of zinc sulfate used to cause formation of the microcarriers.

Microcarriers

The term microcarriers as used herein refers to carriers having a diameter measured in micrometers as well as nanometers. Preferably, the microparticles described herein have a diameter between 5 nm and 1000 microns. A more preferred range of the diameter of the microcarriers is between 50 nm and 30 microns. The term microcarriers refers to solid microparticles as well as hollow microspheres and microgranules. The microcarriers can be spherical or have other shapes. The size of the microcarriers can be varied to suit the appropriate use, such as, for example intravenous, oral or pulmonary delivery. Microcarriers having a diameter of at least fifteen microns are useful for regional or depot delivery, whereas delivery via inhalation requires smaller particles in the one to five micron size. Intravenous administration generally requires nanoparticles, in the 20 to 300 nm range, preferably 50 to 150 nm.

Other Excipients

Excipients such as polyalcohols can be used to stabilize the biomolecule. Examples are mannitol and trehalose. Surfactants can also be used to add stability. Examples include Tween.RTM., cationic detergents such as cetyltrimethyl-ammonium chloride, and anionic phospholipids such as diolylphosphatidylglycerol.

METHODS OF MAKING MICROPARTICLE COMPOSITIONS

The microparticle compositions described above are generally prepared by incorporating the biomolecule within a complex formed by the cationic polymer and the anionic polymer. Preferably, the biomolecule is itself ionically bound to one of the polymers. Alternatively, the biomolecule may be merely physically surrounded by the polymeric complex.

Biomolecules that are positively charged, such as some peptides, can be first reacted with the anionic polymer in solution. The cationic polymer is then added and mixed with the biomolecule/anionic polymer complex. Other biomolecules, such as DNA are reacted first with the cationic polymer and the anionic polymer is then added to the reaction mixture. A catalyst may also be added to enhance the reaction. Preferably, the catalyst is combined with the second polymer that is added to the reaction mixture.

Microcarrier formation is initiated by causing precipitation of the complex. An acid, preferably a dilute acid such as acetic acid or hydrochloric acid, can be added to lower the pH to the point where the reaction becomes cloudy. In most cases, this will be a pH of approximately 5 to 8. Alternatively, salts, such as zinc sulfate, can be added to the solution until the solution becomes cloudy. Certain amino acids that bring the pH into the range of microparticle formation may alternatively be used.

Biomolecules that are negatively charged such as plasmid DNA, can be first reacted with the positively charged polymer, such as polyethyleneimine or polylysine. The anionic polymer is then added to the complex of biomolecule and cationic polymer, whereupon it complexes with the cationic polymer. Again, as the complex is precipitated by lowering the pH is lowered or adding zinc sulfate as described above, microcarriers are formed.

In another embodiment, the anionic and cationic polymers and the biomolecule can be combined to form a polymeric complex entrapping the biomolecule. The complex is then precipitated into microcarriers using a precipitation agent, such as zinc sulfate.

Unlike many conventional particle formation methods, the microparticles described herein are formed in the absence of heat. Therefore, preferably both the polymer complex formation step and the precipitation step are conducted at a temperature between 4^oC. and room temperature. The absence of adverse reaction conditions, such as elevated temperatures, enhances the bioactivity or immunogenicity of the biomolecule incorporated in the microparticle.

The polymers are preferably provided in a concentration of about 0.1 to 20 weight percent, most preferably about 1 weight percent. The polymers are mixed in a weight ratio of 1:9 to 9:1, preferably about 1:4 to 4:1, most preferably about 1:1. The polymers are mixed in a charge ratio of 1:3 to 3:1, most preferably about 1:1. The ratio to be used is a function of the particle size and drug loading capacity desired.

The amount of biomolecule present greatly depends upon the dosage desired for the biomolecule and the degree of interaction between the biomolecule and one or more of the polymers. A highly charged biomolecule can be retained by the polymeric complex in much higher concentration than a relatively uncharged biomolecule. Preferably, biomolecules are incorporated into the microparticle with an efficiency of 80 to 90%. One skilled in the art can determine appropriate amounts of biomolecule.

The size of the microcarriers can be controlled by the concentration of the polymers, the charge ratio of the polymers, the molecular weight of the polymers, and the pH of the reaction. The ability to control microparticle size is especially important for administration of the biomolecule to a human or animal subject. As described above, particular diameter ranges are required for certain routes of administration, such as the need for nanoparticles when intravenous administration is employed.

The microparticles are separated from the non-incorporated components of the incubation mixture by conventional separation methods well known to those skilled in the art such as centrifugation, filtration and sedimentation. Preferably, the reaction mixture is centrifuged so that the microparticles sediment to the bottom of the centrifuge tube and the non-incorporated components remain in the supernatant, which is then removed by decanting. Alternatively, a suspension containing formed microparticles is filtered so that the microparticles are retained on the filter and the non-incorporated components pass through the filter.

Further purification of the microparticles is achieved by washing in an appropriate volume of a washing solution. The preferred washing solution is a buffer, most preferably a nonionic aqueous solution or a nonionic aqueous solution containing water soluble polymers. Repeated washings can be utilized as necessary and the microparticles separated from the wash solution as described above.

The final microparticle composition may be lyophilized, stored as a wet cake, or formulated in solution for final use.

Microparticle Post-production Treatment

The microparticles are optionally treated after production to enhance or impart particular characteristics to the microparticles, such as stability, detectability, and prolonged release. For example, vanillin can be added to stabilize the biomolecule and help prolong its release by rendering the biomolecules less soluble the matrix.

The microparticles are optionally labelled with a detectable label using various types of labels and methods of labelling molecules well known to those skilled in the art. For example, the label can be a metal, a radiolabel, or a Mass or Nuclear Magnetic Resonance (NMR) label. Dyes, chemiluminescent agents, bioluminescent agents and fluorogens can also be used to label the microparticles. The microparticles can also be labelled with a chromogen, or enzyme to produce a chromogenic or fluorogenic reaction upon addition of substrate. Alternatively, the microparticles can be biotinylated and utilized in a biotin-avidin reaction, which may also be coupled to a label such as an enzyme or fluorogen. A label can also be made by incorporating any modified base, amino acid, or precursor containing any label, incorporation of a modified base of amino acid containing a chemical group recognizable by specific antibodies, or by detecting any bound antibody complex by various means including immunofluorescence or immuno-enzymatic reactions. Such labels can be detected using enzyme-linked immunoassays (ELISA) or by detecting a color change with the aid of a spectrophotometer.

Molecules may be attached to the outer surface of the microparticles by methods known to those skilled in the art to "coat" the microparticles. These molecules are attached for purposes such as to enhance stability and facilitate targeting. For example, biomolecules such as phospholipids may be attached to the surface of the microparticle to prevent endocytosis by endosomes; receptors, antibodies or hormones may be attached to the surface to promote or facilitate targeting of the microparticle to the desired organ, tissue or cells of the body; and polysaccharides, such as glucans, may be attached to the outer surface of the microparticle to enhance or to avoid uptake by macrophages. The microparticles may also be coated with one or more stabilizing substances, which may be particularly useful for long term depoting with parenteral administration or for oral delivery by allowing passage of the microparticles through the stomach or gut without dissolution. For example, microparticles intended for oral delivery may be stabilized with a coating of a substance such as mucin. Additionally, the particles can be non-covalently coated with compounds such as fatty acids or lipids. The coating may be applied to the microparticles by immersion in the solubilized coating substance, spraying the microparticles with the substance or other methods well known to those skilled in the art.

METHODS OF USING MICROPARTICLE COMPOSITIONS

The compositions are useful for producing microcarriers for use in the delivery of the complexed biomolecule. The biomolecule is useful for achieving a therapeutic, diagnostic or prophylactic effect. The microcarriers may be used for oral or pulmonary delivery of a biomolecule or via intravenous injection. The microcarriers are also useful in methods involving transdermal drug delivery as well as subcutaneous and intramuscular delivery.

A particular use of the microcarriers is in vaccine formulation. Microparticles containing antigens capable of provoking an immune response as the biomolecules are particularly suitable for use as vaccines. It has been unexpectedly discovered that the polymers of the microparticles described herein, particularly polyethyleneimine and dextran sulfate, have adjuvant effects. Therefore, the microparticles produce an immunogenic effect when administered to a human or animal subject in the absence of a conventional adjuvant, such as aluminum hydroxide (alum).

Claim 1 of 15 Claims

What is claimed is:

1. A microparticle composition for delivery of a biomolecule comprising a biomolecule incorporated in a complex comprising a cationic polymer ionically complexed with an anionic polymer, wherein the cationic polymer is polyethyleneimine and the anionic polymer is dextran sulfate.

____________________________________________
If you want to learn more about this patent, please go directly to the U.S. Patent and Trademark Office Web site to access the full patent.