US20170112776A1

US20170112776A1 - Coated controlled release polymer particles as efficient oral delivery vehicles for biopharmaceuticals

Info

Publication number: US20170112776A1
Application number: US15/349,681
Authority: US
Inventors: Sangyong Jon; Omid C. Farokhzad; Robert S. Langer; Jianjun Cheng
Original assignee: Brigham and Womens Hospital Inc; Massachusetts Institute of Technology
Current assignee: Brigham and Womens Hospital Inc; Massachusetts Institute of Technology
Priority date: 2004-11-04
Filing date: 2016-11-11
Publication date: 2017-04-27
Also published as: WO2007001448A2; WO2007001448A3; US9492400B2; US20080268063A1

Abstract

A composition for delivering an active agent to a patient. The composition includes a polymer core encapsulating the active agent and a mucoadhesive coating disposed about the core. The polymer may include covalently linked poly(ethylene glycol) chains, and the mucoadhesive coating may be selected to facilitate transfer of the particle through the intestinal mucosa. A molecular weight and cross-link density of the polymer may be selected such that the polymer core will decompose in a predetermined time interval. The fraction of the dose of the drug entering the system at circulation during the predetermined time interval may be between about 0.25% and about 25%. The composition may be formulated as a plurality of nanoparticles or microparticles that are combined with a pharmaceutically acceptable carrier to produce an edible or inhalable drug product.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. Ser. No. 11/666,908, filed Feb. 26, 2008, which claims priority to International Patent Application No. PCT/US2005/040100 entitled “Coated Controlled Release Polymer Particles As Efficient Oral Delivery Vehicles or Biopharmaceuticals,” filed Nov. 4, 2005, which claims the priority of U.S. Provisional Application No. 60/625,001, filed Nov. 4, 2004, the entire contents of which are incorporated herein by reference.

FIELD OF THE INVENTION

This invention pertains to drug delivery vehicles, and, more particularly, to controlled release particles coated with a mucoadhesive material.

BACKGROUND OF THE INVENTION

A vast pharmacopeia is available to treat conditions ranging from the annoyance of dry skin to life-threatening diseases. Many of these remedies can be administered orally, either through ingestion or inhalation, or through the skin as a patch or ointment. Others are susceptible to enzymatic degradation by proteases and other chemicals in the gastrointestinal (GI) tract or exhibit poor permeability through the skin or intestinal epithelial cells (enterocytes). Such drugs must be administered through less convenient methods, for example, by injection.
Unless a pharmaceutical is administered continuously, for example, using an intravenous drip, the serum levels of the drug will not be continuous. Serum levels will spike shortly after administration and then tail off in a non-linear fashion. While there may be an optimal serum concentration, a patient will only experience this optimum concentration briefly, as the concentration of the drug decreases from the initial spike. While the average concentration over time may be correct, the actual serum concentration of the drug will practically always be greater or less than optimal.
Another factor that tends to impede a patient's receipt of the proper quantity of a drug is patient compliance. Many patients are unwilling or unable to comply with a physician's instructions describing how often to take a drug. It is inconvenient and confusing to take several drugs at different times during the day and painful to inject protein drugs such as insulin.
The use of controlled-release formulations to provide a consistent dose of a drug to a patient has been an active area of research for decades and has been fueled by the many recent developments in polymer science and the need to deliver more labile pharmaceutical agents such as nucleic acids, proteins, and peptides. Controlled release polymer systems can be designed to provide a drug level in the optimum range over a longer period of time than other drug delivery methods, thus increasing both the efficacy of the drug and patient compliance.
Biodegradable particles have been developed as sustained release vehicles used in the administration of small molecule drugs as well as protein and peptide drugs and nucleic acids (Langer, Science, 249:1527-1533, 1990; Mulligan, Science, 260:926-932, 1993; Eldridge, Mol. Immunol., 28:287-294, 1991; the entire teaching of each of the foregoing references is incorporated herein by reference). The drugs are typically encapsulated in a polymer matrix which is biodegradable and biocompatible. As the polymer is degraded or dissolved and/or as the drug diffuses out of the polymer, the drug is released into the body. Typically, polymers used in preparing these particles are polyesters such as poly(glycolide-co-lactide) (PLGA), polyglycolic acid, poly-β-hydroxybutyrate, and polyacrylic acid ester. These particles have the additional advantage of protecting the drug from degradation by the body.
Still, it is desirable to have controlled-release system that can be used for oral administration of substances that are not normally stable in the gastrointestinal tract or that are difficult to transport across the intestinal mucosa into the bloodstream. Oral delivery is expected to result in enhanced patient compliance, resulting in improved clinical outcomes, largely due to ease of drug administration as compared to subcutaneous or intravenous injection. An appropriate delivery system that can 1) encapsulate protein and other labile drugs, 2) protect the drugs while in transit through the gastrointestinal (GI) tract, 3) efficiently transport the drugs across the intestinal mucosa, and 4) efficiently release the drugs in the systemic circulation may result in high bioavailability of protein drugs after oral administration. Even for drugs that are stable in the GI tract, a delivery system that can transport them across the intestinal mucosa and release them directly into the bloodstream can enhance bioavailability.

SUMMARY OF THE INVENTION

In one aspect, the invention is a composition for delivering an active agent to a patient. The composition includes a polymer core encapsulating a predetermined amount of the active agent and a mucoadhesive coating disposed about the core to form a coated particle. The polymer includes covalently linked poly(alkylene glycol) chains. The mucoadhesive coating is retained on the core through one or more of covalent interactions, electrostatic interactions, affinity interactions, metal coordination, physical adsorption, host-guest interactions, and hydrogen bonding interactions. A molecular weight and cross-link density of the biodegradable polymer is selected such that the polymer core will decompose over a predetermined time interval. The mucoadhesive coating is selected to facilitate transfer of the particle through the intestinal mucosa. The fraction of the predetermined amount entering the systemic circulation during the predetermined time interval is between about 0.25% and about 25%, for example, between about 5% and about 20% or between about 10% and about 15%.
The composition may further include a targeting agent disposed under the mucoadhesive coating and, optionally, an intermediate layer disposed between the targeting agent and the mucoadhesive coating. The intermediate layer may include a first material while the mucoadhesive coating includes a second material, and the first material and the second material may have opposing electrostatic charges at pH 2, but not at pH 7.4. The intermediate layer may include a biodegradable polymer, and the targeting agent may be disposed at a surface portion of the polymer core, an interior portion of the polymer core, or both. The targeting agent may be one or more of nucleic acid aptamers, growth factors, hormones, cytokines, interleukins, antibodies, integrins, fibronectin receptors, p-glycoprotein receptors, and cell binding sequences, for example, RGD.
The core may include PEGylated poly (lactic acid). The coating may be a block co-polymer having a mucoadhesive block and a block that is adapted to participate in an interaction selected from electrostatic interactions, affinity interactions, metal coordination, physical adsorption, host-guest interactions, and hydrogen bonding interactions. The active agent may be a biomolecule, bioactive agent, small molecule, drug, protein, vaccine, or polynucleotide.
The poly(alkylene glycol) may be carboxylated and may be selected from poly(ethylene glycol) and poly(propylene glycol). The poly(alkylene glycol) may have a molecular weight between about 100 and about 7000 Daltons, for example, between about 100 and about 1000 Daltons, between about 1000 Daltons and 3500 Daltons, between 3500 Daltons and about 7000 Daltons, or more.
The coating may include one or more of chitosan, poly(lysine), poly(ethylene imine), lecithin, lectin, polycarboxylic acids, poly(acrylic acids), polysaccharides, hydrogels, monosaccharides, oligosaccharides, oligopeptides, polypeptides, and co-polymers of these.
In another aspect, the invention is a composition for administering an active agent to a patient. The composition includes a plurality of particles, each particle including a polymer core encapsulating the active agent and a mucoadhesive coating disposed about the core to form a coated particle, and a pharmaceutically acceptable carrier combined with the plurality of particles. The pharmaceutically acceptable carrier is edible or inhalable.
In another aspect, the invention is a method for administering an active agent to a patient. The method includes orally administering to the patient a composition comprising a plurality of particles. Each particle includes a polymer core encapsulating the active agent and a mucoadhesive coating disposed about the core to form a coated particle. The composition further includes a pharmaceutically acceptable edible carrier.

DEFINITIONS

“Bioavailability”: The term “bioavailability”, as used herein, refers to the rate at which and extent to which an active agent is absorbed or is otherwise available to a treatment site in the body. For active agents that are encapsulated in a biodegradable polymer or pharmaceutically acceptable carrier, or both, bioavailability also depends on the extent to which the active agent is released from the polymer and/or carrier into the bloodstream.
“Biomolecules”: The term “biomolecules”, as used herein, refers to molecules (e.g., proteins, amino acids, peptides, polynucleotides, nucleotides, carbohydrates, sugars, lipids, nucleoproteins, glycoproteins, lipoproteins, steroids, etc.) whether naturally-occurring or artificially created (e.g., by synthetic or recombinant methods) that are commonly found in cells and tissues. Specific classes of biomolecules include, but are not limited to, enzymes, receptors, neurotransmitters, hormones, cytokines, cell response modifiers such as growth factors and chemotactic factors, antibodies, vaccines, haptens, toxins, interferons, ribozymes, anti-sense agents, plasmids, DNA, and RNA.
“Biocompatible”: The term “biocompatible”, as used herein is intended to describe compounds that are not toxic to cells. Compounds are “biocompatible” if their addition to cells in vitro results in less than or equal to 20% cell death, and their administration in vivo does not induce significant inflammation or other such significant adverse effects.
“Biodegradable”: As used herein, “biodegradable” polymers are polymers that degrade fully (i.e., down to monomeric species) under physiological or endosomal conditions. In preferred embodiments, the polymers and polymer biodegradation byproducts are biocompatible. Biodegradable polymers are not necessarily hydrolytically degradable and may require enzymatic action to fully degrade.
“Decomposition”: As used herein, “decomposition” is the process by which a material is broken down under physiological conditions into components that may be metabolized by the body. For example, biodegradable polymers are degraded to monomeric species. Non-biodegradable polymers may be dissolved and removed from the bloodstream by the kidneys. Alternatively, the material or its components may be metabolized by the liver.
“Endosomal conditions”: The phrase “endosomal conditions”, as used herein, relates to the range of chemical (e.g., pH, ionic strength) and biochemical (e.g., enzyme concentrations) conditions likely to be encountered within endosomal vesicles. For most endosomal vesicles, the endosomal pH ranges from about 5.0 to 6.5.
“Physiological conditions”: The phrase “physiological conditions”, as used herein, relates to the range of chemical (e.g., pH, ionic strength) and biochemical (e.g., enzyme concentrations) conditions likely to be encountered in the intracellular and extracellular fluids of tissues. For most tissues, the physiological pH ranges from about 7.0 to 7.4.
“Polynucleotide”, “nucleic acid”, or “oligonucleotide”: The terms “polynucleotide”, “nucleic acid”, or “oligonucleotide” refer to a polymer of nucleotides. The terms “polynucleotide”, “nucleic acid”, and “oligonucleotide”, may be used interchangeably. Typically, a polynucleotide comprises at least two nucleotides. DNAs and RNAs are polynucleotides. The polymer may include natural nucleosides (i.e., adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine, deoxyguanosine, and deoxycytidine), nucleoside analogs (e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine, 3-methyl adenosine, C5-propynylcytidine, C5-propynyluridine, C5 bromouridine, C5 fluorouridine, C5 iodouridine, C5 methylcytidine, 7 deazaadenosine, 7 deazaguanosine, 8 oxoadenosine, 8 oxoguanosine, O(6) methylguanine, and 2-thiocytidine), chemically modified bases, biologically modified bases (e.g., methylated bases), intercalated bases, modified sugars (e.g., 2′-fluororibose, ribose, 2′-deoxyribose, arabinose, and hexose), or modified phosphate groups (e.g., phosphorothioates and 5′-N phosphoramidite linkages). Enantiomers of natural or modified nucleosides may also be used. Nucleic acids also include nucleic acid-based therapeutic agents, for example, nucleic acid ligands, siRNA, short hairpin RNA, antisense oligonucleotides, ribozymes, aptamers, and SPIEGELMERS™, oligonucleotide ligands described in Wlotzka, et al., Proc. Nat'l. Acad. Sci. USA, 2002, 99(13):8898, the entire contents of which are incorporated herein by reference.
“Polypeptide”, “peptide”, or “protein”: According to the present invention, a “polypeptide”, “peptide”, or “protein” comprises a string of at least three amino acids linked together by peptide bonds. The terms “polypeptide”, “peptide”, and “protein”, may be used interchangeably. Peptide may refer to an individual peptide or a collection of peptides. Inventive peptides preferably contain only natural amino acids, although non natural amino acids (i.e., compounds that do not occur in nature but that can be incorporated into a polypeptide chain) and/or amino acid analogs as are known in the art may alternatively be employed. Also, one or more of the amino acids in a peptide may be modified, for example, by the addition of a chemical entity such as a carbohydrate group, a phosphate group, a farnesyl group, an isofarnesyl group, a fatty acid group, a linker for conjugation, functionalization, or other modification, etc. In one embodiment, the modifications of the peptide lead to a more stable peptide (e.g., greater half-life in vivo). These modifications may include cyclization of the peptide, the incorporation of D-amino acids, etc. None of the modifications should substantially interfere with the desired biological activity of the peptide.
“Polysaccharide”, “carbohydrate” or “oligosaccharide”: The terms “polysaccharide”, “carbohydrate”, or “oligosaccharide” refer to a polymer of sugars. The terms “polysaccharide”, “carbohydrate”, and “oligosaccharide”, may be used interchangeably. Typically, a polysaccharide comprises at least two sugars. The polymer may include natural sugars (e.g., glucose, fructose, galactose, mannose, arabinose, ribose, and xylose) and/or modified sugars (e.g., 2′-fluororibose, 2′-deoxyribose, and hexose).
“Mucoadhesive”: As used herein, the term “mucoadhesive” is used to indicate that a moiety has an affinity for a component of the intestinal wall. The affinity may be specific, for example, a specific affinity for a protein or sugar found in the membrane of a cell, for example, an M cell or intestinal epithelial cell, or non-specific, for example, a tendency to non-covalently bind to the mucosa.
“Small molecule”: As used herein, the term “small molecule” is used to refer to molecules, whether naturally-occurring or artificially created (e.g., via chemical synthesis) that have a relatively low molecular weight. In some embodiments, small molecules are monomeric and have a molecular weight of less than about 1500 g/mol. Preferred small molecules are biologically active in that they produce a local or systemic effect in animals, preferably mammals, more preferably humans. Small molecules include, but are not limited to, radionuclides and imaging agents. In certain embodiments, the small molecule is a drug. Preferably, though not necessarily, the drug is one that has already been deemed safe and effective for use by the appropriate governmental agency or body. For example, drugs for human use listed by the FDA under 21 C.F.R. §§330.5, 331 through 361, and 440 through 460; drugs for veterinary use listed by the FDA under 21 C.F.R. §§500 through 589, incorporated herein by reference, are all considered acceptable for use in accordance with the present invention. Known naturally-occurring small molecules include, but are not limited to, penicillin, erythromycin, taxol, cyclosporin, and rapamycin. Known synthetic small molecules include, but are not limited to, ampicillin, methicillin, sulfamethoxazole, and sulfonamides.
“Bioactive agents”: As used herein, “bioactive agents” is used to refer to compounds or entities that alter, inhibit, activate, or otherwise affect biological or chemical events. For example, bioactive agents may include, but are not limited to, anti-AIDS substances, anti-cancer substances, antibiotics, immunosuppressants, anti-viral substances, enzyme inhibitors, including but not limited to protease and reverse transcriptase inhibitors, fusion inhibitors, neurotoxins, opioids, hypnotics, anti-histamines, lubricants, tranquilizers, anti-convulsants, muscle relaxants and anti-Parkinson substances, anti-spasmodics and muscle contractants including channel blockers, miotics and anti-cholinergics, anti-glaucoma compounds, anti-parasite and/or anti-protozoal compounds, modulators of cell-extracellular matrix interactions including cell growth inhibitors and anti-adhesion molecules, vasodilating agents, inhibitors of DNA, RNA or protein synthesis, anti-hypertensives, analgesics, anti-pyretics, steroidal and non-steroidal anti-inflammatory agents, anti-angiogenic factors, anti-secretory factors, anticoagulants and/or antithrombotic agents, local anesthetics, ophthalmics, prostaglandins, anti-depressants, anti-psychotic substances, anti-emetics, and imaging agents. In a certain embodiments, the bioactive agent is a drug.
A more complete listing of bioactive agents and specific drugs suitable for use in the present invention may be found in “Pharmaceutical Substances: Syntheses, Patents, Applications” by Axel Kleemann and Jurgen Engel, Thieme Medical Publishing, 1999; the “Merck Index: An Encyclopedia of Chemicals, Drugs, and Biologicals”, Edited by Susan Budavari et al., CRC Press, 1996, and the United States Pharmacopeia-25/National Formulary-20, published by the United States Pharmcopeial Convention, Inc., Rockville Md., 2001, all of which are incorporated herein by reference.
As used herein, the term “pharmaceutically active agent” refers collectively to biomolecules, small molecules, and bioactive agents.

BRIEF DESCRIPTION OF THE DRAWING

The invention is described with reference to the several figures of the drawing, in which,

FIG. 1 is a schematic diagram of a particle according to an embodiment of the invention.

FIGS. 2A&B are graphs showing the transport yields of nanoparticles (A) and micorparticles (B) across monolayers of Caco-2 cells with and without a mucoadhesive coating.

FIGS. 3A&B are graphs showing the absorption yields of nanoparticles (A) and micorparticles (B) into mice.

FIG. 4A is a graph showing the whole blood absorption of ¹²⁵I-insulin delivered to balb/C mice by PEGylated and non-PEGylated PLA nanoparticles.

FIG. 4B is a graph comparing the whole blood absorption of ¹²⁵I-insulin delivered to balb/C mice by PEGylated PLA nanoparticles and microparticles

FIG. 5 is a graph illustrating the decrease in plasma glucose over time after administration of insulin encapsulated in PLA-PEG-COOH and PLGA-PEG-COOH nanoparticles.

FIG. 6 is a graph illustrating the reduction in plasma glucose after intravenous administration of Humulin R (0.5 U/kg)

FIG. 7 is a graph illustrating the linear relationship between absorbance and plasma insulin concentration

FIG. 8 is a graph illustrating the serum insulin concentration with respect to time after intravenous and intraperitoneal administration of Humulin-R

FIG. 9 is a graph illustrating the serum insulin concentration with respect to time after administration of encapsulated Humulin with various surface coatings.

DETAILED DESCRIPTION OF CERTAIN PREFERRED EMBODIMENTS

A delivery device for biopharmaceuticals and other substances that are not easily transported from the gastrointestinal tract to the circulatory system or that are not stable in the gastrointestinal tract includes a polymer encapsulating an active agent and a mucoadhesive surface.
In one embodiment, the device is a plurality of microparticles or nanoparticles 10 having a core 12 in which a pharmaceutical is encapsulated by a controlled-release polymer and a mucoadhesive coating 14 disposed about the core (FIG. 1). The controlled-release polymer may be biodegradable, and the coating may be biodegradable as well. The coating is affixed to the core by covalent or non-covalent interactions. The coating may remain in place as the core material decomposes. Such particles may be ingested by a patient. The particles may be nanoparticles, having a size between about 10 and about 1000 nm, for example, between about 10 and about 100 nm, between 100 and about 500 nm, or between about 500 and about 1000 nm. Alternatively, the particles may be microparticles having a size between about 1 and about 100 micrometers, for example, between about 1 and about 10 micrometers, between about 10 and about 50 micrometers, or between about 50 and about 100 micrometers. The particles adhere to the intestinal lining and gradually pass through the lining into the circulatory system, where they gradually release the active agent at a rate determined by the decomposition rate of the core. Alternatively, the core material may also include a mucoadhesive, obviating a coating.

Encapsulation Materials

Materials for use in encapsulating pharmaceuticals for use with the invention may be biodegradable. A variety of biodegradable polymers are well known to those skilled in the art. Exemplary synthetic polymers suitable for use with the invention include but are not limited to poly(arylates), poly(anhydrides), poly(hydroxy acids), polyesters, poly(ortho esters), polycarbonates, poly(propylene fumerates), poly(caprolactones), polyamides, polyphosphazenes, polyamino acids, polyethers, polyacetals, polylactides, polyhydroxyalkanoates, polyglycolides, polyketals, polyesteramides, poly(dioxanones), polyhydroxybutyrates, polyhydroxyvalyrates, polycarbonates, polyorthocarbonates, poly(vinyl pyrrolidone), biodegradable polycyanoacrylates, polyalkylene oxalates, polyalkylene succinates, poly(malic acid), poly(methyl vinyl ether), poly(ethylene imine), poly(acrylic acid), poly(maleic anhydride), biodegradable polyurethanes and polysaccharides. U.S. patents that describe the use of polyanhydrides for controlled delivery of substances include U.S. Pat. No. 4,857,311 to Domb and Langer, U.S. Pat. No. 4,888,176 to Langer, et al., and U.S. Pat. No. 4,789,724 to Domb and Langer.
Naturally-occurring polymers, such as polysaccharides and proteins, may also be employed. Exemplary polysaccharides include alginate, starches, dextrans, celluloses, chitin, chitosan, hyaluronic acid and its derivatives; exemplary proteins include collagen, albumin, and gelatin. Polysaccharides such as starches, dextrans, and celluloses may be unmodified or may be modified physically or chemically to affect one or more of their properties such as their characteristics in the hydrated state, their solubility, or their half-life in vivo.
In other embodiments, the polymer includes polyhydroxy acids such as polylactic acid (PLA), polyglycolic acid (PGA), their copolymers poly(lactic-co-glycolic acid) (PLGA), and mixtures of any of these. These polymers are among the synthetic polymers approved for human clinical use as surgical suture materials and in controlled release devices. They are degraded by hydrolysis to products that can be metabolized and excreted. Furthermore, copolymerization of PLA and PGA offers the advantage of a large spectrum of degradation rates from a few days to several years by simply varying the copolymer ratio of glycolic acid to lactic acid, which is more hydrophobic and less crystalline than PGA and degrades at a slower rate.
Non-biodegradable polymers may also be employed for use with the invention. Exemplary non-biodegradable, yet biocompatible polymers include polystyrene, polyesters, non-biodegradable polyurethanes, polyureas, poly(vinyl alcohol), polyamides, poly(tetrafluoroethylene), poly(ethylene vinyl acetate), polypropylene, polyacrylate, non-biodegradable polycyanoacrylates, non-biodegradable polyurethanes, polymethacrylate, poly(methyl methacrylate), polyethylene, polypyrrole, polyanilines, polythiophene, and poly(ethylene oxide).
Any of the above polymers may be functionalized with a poly(alkylene glycol), for example, poly(ethylene glycol) (PEG) or poly (propyleneglycol) (PPG), or may have a particular terminal functional group, e.g., poly(lactic acid) modified to have a terminal carboxyl group. Exemplary PEGylated polymers include but are not limited to PEGylated poly(lactic acid), PEGylated poly(lactic-co-glycolic acid), PEGylated poly(caprolactone), PEGylated poly(ortho esters), PEGylated polylysine, and PEGylated poly(ethylene imine). Poly(alkylene glycols) are known to increase the bioavailability of many pharmacologically useful compounds, partly by increasing the gastrointestinal stability of derivatized compounds. Poly(alkylene glycols) chains may be as short as about 100 Daltons or have a molecular weight of about 1000, about 3000, about 5000, about 7000 Daltons, or more. The poly(alkylene glycol) chain may also be modified to have a charged endgroup or other group selected to engage in a particular interaction with the coating material. For example, carboxylated PEG will engage in electrostatic interactions with positively charged coating materials such as chitosan.
Co-polymers, mixtures, and adducts of any of the above modified and unmodified polymers may also be employed. For example, amphiphilic block co-polymers having hydrophobic regions and anionic or otherwise hydrophilic regions may be employed. Block co-polymers having regions that engage in different types of non-covalent or covalent interactions may also be employed. For example, a block co-polymer may have one block that is optimized to interact with an active agent being encapsulated and another block optimized to interact with the bioadhesive coating (see below). Alternatively or in addition, polymers may be chemically modified to have particular functional groups. For example, polymers may be functionalized with hydroxyl, amine, carboxy, maleimide, thiol, N-hydroxy-succinimide (NHS) esters, or azide groups. These groups may be used to render the polymer hydrophilic or to achieve particular interactions with coating materials as described below.
One skilled in the art will recognize that the molecular weight and the degree of cross-linking may be adjusted to control the decomposition rate of the polymer and thus the release rate of the pharmaceutical. Methods of controlling molecular weight and cross-linking to adjust release rates are well known to those skilled in the art.
A variety of methods of making particles in which active agents are encapsulated are well known to those skilled in the art. For example, a double emulsion technique may be used to combine a polymer and active agent in particles. Alternatively, particles may be prepared by spray-drying.

Coating Materials

Positively charged biocompatible materials such as chitosan, poly(L-lysine), and poly(ethylene imines) are suitable for coating particles for use with the invention. Lectins may also be used to coat particles. Lectins may particularly target M cells in Peyer's patches in the intestine, enhancing the affinity of the particles for the intestinal wall. Lectins are produced by a wide variety of plants; one skilled in the art will recognize that not all lectins are appropriate for use in pharmaceutical compositions. A wide variety of lectins are available from Sigma-Aldrich, which also provides information on the toxicity and mutagenicity of commercially available lectins. One skilled in the art will recognize that lectins that are found in commonly eaten foods are more likely to be suitable for use with embodiments of the invention. Negatively charged materials may also be employed. Exemplary bioadhesive materials also include, without limitation, lecithin, polycarboxylic acids, poly(acrylic acids), polysaccharides, monosaccharides, oligosaccharides, oligopeptides, polypeptides, and co-polymers of two or more mucoadhesive materials. Alternatively or in addition, mucoadhesive or non-mucoadhesive polymers may be modified with mucoadhesive materials. For example, sugars may be covalently linked to polyacrylates. Polymers having regions adapted to bind the coating to the core material and regions adapted to be mucoadhesive may also be employed. For example, a block co-polymer of a polycation and a hydrogen bond donor can be used to coat a core containing a polymer that acts as a hydrogen bond receptor. Additional bioadhesive molecules that may be used with the invention include but are not limited to hydrophilic and amphiphilic polymers, hydrogels, and the polymers disclosed in U.S. Pat. Nos. 6,217,908, 6,297,337; 6,514,535; and 6,284,235 the contents of which are incorporated herein by reference. Bioadhesive molecules may be PEGylated or otherwise modified as described above.
One skilled in the art will recognize that excessive cross-linking of the coating material may hinder release of the active agent from the core of the particle. The skilled artisan will also recognize that the effect of cross-linking may be easily tested by measuring the release of an active agent or a labeled analog from particles coated with materials having different degrees of cross-linking.
In another embodiment, the particles are provided with a double coating. For example, the particles may include a targeting agent that helps direct the particles to a specific tissue once they enter the blood stream. Exemplary targeting agents include nucleic acid aptamers, growth factors, hormones, cytokines, interleukins, antibodies, integrins, fibronectin receptors, p-glycoprotein receptors, and cell binding sequences such as RGD. Nucleic acid aptamers selective for a particular target may be known from the literature or may be identified using any method known to those skilled in the art, for example, the methods disclosed in U.S. Pat. Nos. 5,270,163, 5,475,096, and 6,114,120, the contents of which are incorporated herein by reference. Aptamers for certain tissues may also be obtained commercially, for example, from Archemix Corp. These targeting agents may be attached to the surface of the particle or may be attached to the polymer itself before the particles are formed. The particles are then coated with a negatively charged material, e.g., a negatively charged polymer. Exemplary polymers include carboxymethylcellulose, polyacrylic acid, polymethacrylic acid, polystyrenesulfonate, and polymers including carboxylate, sulfonate, sulfate, phosphate, or nitrate groups. A positively charged mucoadhesive material is then coated over the negatively charged material.
After the particle crosses the intestinal wall into the bloodstream, the environmental pH increases from about 2-3 to about 7.4. Depending on the pKa of the negative coating, it may become neutrally charged, reducing its affinity for the positively charged mucoadhesive coating. As a result, the mucoadhesive coating becomes dislodged from the particle. The negatively charged coating may also be biodegradable, for example, through hydrolysis or enzymatic mechanisms. In this embodiment, whether the pKa of the negatively charged coating is such that it will become neutrally charged after entering the bloodstream, the degradation of the coating will dislodge the outer mucoadhesive coating from the particle. In any of these embodiments, the two coatings protect both the agent being delivered and the targeting agent from degradation in the digestive system while allowing the targeting agent to be exposed at the surface of the particles after they enter the bloodstream.

Pharmaceutical Compositions

The active agents to be incorporated in the controlled release polymer system of the present invention may be therapeutic, diagnostic, prophylactic or prognostic agents. Any chemical compound to be administered to an individual may be delivered using the conjugates of the invention. The active agent may be a small molecule, organometallic compound, nucleic acid, protein, peptide, metal, an isotopically labeled chemical compound, drug, vaccine, immunological agent, etc. Exemplary active agents include small molecules, biomolecules, and bioactive agents as defined herein.
In one embodiment, the agents are organic compounds with pharmaceutical activity. In another embodiment of the invention, the agent is a small molecule that is a clinically used drug. In exemplary embodiments, the drug is an antibiotic, anti-viral agent, anesthetic, steroidal agent, anti-inflammatory agent, anti-neoplastic agent, antigen, vaccine, antibody, decongestant, antihypertensive, sedative, birth control agent, progestational agent, anti-cholinergic, analgesic, anti-depressant, anti-psychotic, adrenergic blocking agent, diuretic, cardiovascular active agent, vasoactive agent, non-steroidal anti-inflammatory agent, nutritional agent, etc. While many small molecule drugs are already available for oral administration, some are not sufficiently soluble to be orally administered and may benefit from the techniques described herein.
In another embodiment, the agent is a protein drug, such as an antibody, an antibody fragment, a recombinant antibody, a recombinant protein, a purified protein, a peptide, an amino acid and combinations thereof. Exemplary protein drugs include but are not limited to biologically active macromolecules such as enzyme inhibitors, colony-stimulating factors, plasminogen activators, polypeptide hormones, insulin, myelin basic protein, collagen S antigen, calcitonin, angiotensin, vasopressin, desmopressin, LH-RH (luteinizing hormone-releasing hormone), somatostatin, glucagon, somatomedin, oxytocin, gastrin, secretin, h-ANP (human atrial natriuretic polypeptide), ACTH (adrenocorticotropic hormone), MSH (melanocyte stimulating hormone), beta-endorphin, muramyl dipeptide, enkephalin, neurotensin, bombesin, VIP (vasoactive intestinal peptide), CCK-8 (cholecystokinin), PTH (parathyroid hormone), CGRP (calcitonin gene related peptide), endothelin, TRH (thyroid releasing hormone), interferons, cytokines, streptokinase, urokinase, and growth factors. Exemplary growth factors include but are not limited to activin A (ACT), retinoic acid (RA), epidermal growth factor, bone morphogenetic protein, platelet derived growth factor, hepatocyte growth factor, insulin-like growth factors (IGF) I and II, hematopoietic growth factors, peptide growth factors, erythropoietin, angiogenic factors, anti-angiogenic factors, interleukins, tumor necrosis factors, interferons, colony stimulating factors, t-PA (tissue plasminogen activator), G-CSF (granulocyte colony stimulating factor), heparin binding growth factor (HBGF), alpha or beta transforming growth factor (α- or β-TGF), fibroblastic growth factors, epidermal growth factor (EGF), vascular endothelium growth factor (VEGF), nerve growth factor (NGF) and muscle morphogenic factor (MMP). Also suitable for use with the invention are recombinantly-produced derivatives of therapeutically useful proteins, including deletion, insertion and substitution variants, which on the whole have similar or comparable pharmacological properties.
Gene therapy technology may also benefit from the techniques of the invention. Genetic material is typically not stable in the GI tract. Polymer encapsulation can protect genetic material and “escort” it through the GI tract and into the bloodstream. In one embodiment, the active agent delivered using the techniques of the invention is a nucleic acid based drug, such as DNA, RNA, modified DNA, modified RNA, antisense oligonucleotides, expression plasmid systems, nucleotides, modified nucleotides, nucleosides, modified nucleosides, nucleic acid ligands (e.g. aptamers), intact genes, a promoter complementary region, a repressor complementary region, an enhancer complementary region, and combinations thereof. A promoter complementary region, a repressor complementary region, or an enhancer complementary region can be fully complementary or partially complementary to the DNA promoter region, repressor region, an enhancer region of a gene for which it is desirable to modulate expression. For example, it may be at least 50% complementary, at least 60% complementary, at least 70% complementary, at least 80% complementary, at least 90% complementary, or at least 95% complementary.
Genetic material is acidic and will form electrostatic bonds with cationic polymers. If it is desirable to avoid strong ionic interactions, nucleic acid based drugs can be encapsulated with anionic polymers or other hydrophilic polymers that do not have cationic groups. For example, polymers modified with short poly(cytosine) tags may be used to encapsulate genetic material. Other examples include but are not limited to polysebacic anhydride (PSA) and poly(lactic acid). These polymers may be modified to carry a more negative charge, for example, a terminal carboxylic acid group can be added to poly(lactic acid).
In another embodiment, the controlled release polymer systems may deliver a diagnostic or prognostic agent used for long term diagnosis of a patient's health. For example, kidney function is determined by delivering an agent, such as creatinine, to the bloodstream that is cleared solely by the glomerulus and then measuring the concentration of the agent in the blood or urine over time. The controlled release particles of the invention can be used to provide a steady state concentration of the clearance agent in the bloodstream for an extended period of time, and periodic assays of the concentration of the agent in the patient's urine can be used to determine the rate of clearance of the agent by the kidneys. Alternative clearance agents, for example, agents that are cleared from the body through other mechanisms, e.g, by the liver or through other metabolic processes, may also be encapsulated and delivered using the controlled release polymer systems described herein.
Prophylactic agents that can be delivered to a patient by exploiting the invention include, but are not limited to, antibiotics and nutritional supplements. For example, the techniques of the invention may be used to deliver nutrients to patients experiencing a deficiency or who are unable to produce or store such substances themselves. For example, vitamin D may be delivered to patients who are unable to synthesize it.
Vaccines and antigens are additional prophylactic agents that may be administered to a patient using the techniques of the invention. Some vaccines require extended exposure to the immune system to stimulate the desired immune response. Micro- or nanoparticles containing a vaccine or antigen may be suspended in a fluid or charged into a capsule and ingested, allowing patients to receive their vaccine orally instead of as an injection. A single administration of a dose of particles produced according to the invention may substitute for multiple injections or reduce the number of administrations. Of course, fast-decomposing particles may be fabricated to encapsulate vaccines that do not require extended exposure. Formulation of the vaccine as a capsule, pill, or ingestible liquid may also improve the shelf life of the vaccine, easing delivery of vaccines to rural or impoverished areas.
Vaccines may comprise isolated proteins or peptides, inactivated organisms and viruses, dead organisms and viruses, genetically altered organisms or viruses, and cell extracts. Prophylactic agents may be combined with interleukins, interferon, cytokines, and adjuvants such as cholera toxin, alum, Freund's adjuvant, etc. Prophylactic agents include antigens of such bacterial organisms as Streptococccus pneumoniae, Haemophilus influenzae, Staphylococcus aureus, Streptococcus pyrogenes, Corynebacterium diphtheriae, Listeria monocytogenes, Bacillus anthracia, Clostridium tetani, Clostridium botulinum, Clostridium perfringens, Neisseria meningitidis, Neisseria gonorrhoeae, Streptococcus mutans, Pseudomonas aeruginosa, Salmonella typhi, Haemophilus parainfluenzae, Bordetella pertussis, Francisella tularensis, Yersinia pestis, Vibrio cholerae, Legionella pneumophila, Mycobacterium tuberculosis, Mycobacterium leprae, Treponema pallidum, Leptospirosis interrogans, Borrelia burgdorferi, Camphylobacter jejuni, and the like; antigens of such viruses as smallpox, influenza A and B, respiratory syncytial virus, parainfluenza, measles, HIV, varicella-zoster, herpes simplex 1 and 2, cytomegalovirus, Epstein-Barr virus, rotavirus, rhinovirus, adenovirus, papillomavirus, poliovirus, mumps, rabies, rubella, coxsackieviruses, equine encephalitis, Japanese encephalitis, yellow fever, Rift Valley fever, hepatitis A, B, C, D, and E virus, and the like; antigens of fungal, protozoan, and parasitic organisms such as Cryptococcus neoformans, Histoplasma capsulatum, Candida albicans, Candida tropicalis, Nocardia asteroides, Rickettsia ricketsii, Rickettsia typhi, Mycoplasma pneumoniae, Chlamydial psittaci, Chlamydial trachomatis, Plasmodium falciparum, Trypanosoma brucei, Entamoeba histolytica, Toxoplasma gondii, Trichomonas vaginalis, Schistosoma mansoni, and the like. These antigens may be in the form of whole killed organisms, peptides, proteins, glycoproteins, carbohydrates, or combinations thereof.
While practically any bioactive agent, small molecule, or drug may benefit from the teachings herein, certain pharmaceutical compositions will find particular utility in the inventive compositions. Proteins such as insulin that are not generally stable in the gastrointestinal system may be encapsulated using the techniques of the invention. For example, diabetics could swallow a capsule containing microparticles or nanoparticles having encapsulated insulin. The particles would adhere to the mucosa and pass through the mucosal layer into the blood stream, where they would gradually release insulin. Peptides and small molecules may be delivered in the same manner. Other biomolecules involved in metabolic disorders may also be delivered using the techniques of the invention. For example, phenylalanine hydroxylase and/or tyrosine may be administered to phenylketonurics. Nutritional and enzymatic supplements may be provided to patients with maple syrup urine disease. The techniques of the invention may be exploited to provided enzyme replacement therapy to treat a host of metabolic diseases including but not limited to Gaucher disease, Fabry disease, Niemann-Pick disease, cystic fibrosis, mucopolysaccharidosis, Tay-Sachs disease, Hurler syndrome, many forms of muscular dystrophy, including Pompe disease, and lysosomal storage disorders (see, for example, Sly, “Enzyme replacement therapy for lysosomal storage disorders: successful transition from concept to clinical practice,” Mo Med. 2004 March-April; 101(2):100-4; Desnick, et al., “Enzyme replacement and enhancement therapies: lessons from lysosomal disorders,” Nat Rev Genet. 2003 February; 4(2):157).
For patients who take a drug every day, the compositions of the invention can reduce the frequency with which patients have to take the drug. For example, a patient could take a pill once a week or once a month instead of daily. In one embodiment, controlled release particles produced using the invention may be used to deliver contraceptive drugs to patients. Instead of taking a pill every day, the formulations of the invention may be used to provide a weekly or monthly dose regimen. Estrogen replacement therapy may be administered in the same manner. For example, female reproductive hormones, for example, estrogen and progesterone, may be formulated as particles using the techniques of the invention.
In one embodiment of the present invention, the agent to be delivered may be a mixture of agents. For example, an antibiotic may be combined with an inhibitor of the enzyme commonly produced by bacteria to inactivate the antibiotic (e.g., penicillin and clavulanic acid). In one embodiment, different active agents may be compounded into particles, and then mixtures of different particles may be combined with a delivery vehicle in specific ratios using the techniques described below to provide different combinations of active agents to patients. For example, cyclic contraceptives work by providing a different ratio of reproductive hormones to patients over the course of three weeks, simulating the manner in which the ratio of estrogen and other hormones vary over the course of a menstrual cycle. Rather than preparing particles with different ratios of estrogen and progesterone, different ratios of particles encapsulating estrogen and progesterone may be compounded into single dosage units.
The techniques of the invention provide improved bioavailability to the compounds delivered thereby. Less of the compound will be lost in the digestive tract than if it were delivered without the protection of the encapsulating material and the PEG tag. The mucoadhesive facilitates increased transfer of the active agent across the intestinal mucosa. Using the techniques of the invention, between 0.25 and 25%, for example, between 10 and 15%, of the active agent delivered in a dosage unit can be made available to the patient through release in the bloodstream. The bioavailability of the active agent may be determined using standard pharmacokinetic techniques known to those skilled in the art. For example, the concentration of the active agent in the bloodstream or of the agent or its derivatives in urine may be measured periodically and used to calculate AUC (area under the curve).

Formation of a Coated Particle

Coatings may be immobilized on the particles using a variety of chemical interactions. For example, positively charged coatings such as chitosan will form electrostatic bonds with negatively charged PLA or PLGA. This interaction prevents the coating from being stripped off the particle as it passes into the bloodstream. Likewise, negatively charged coatings may be employed with positively charged cores.
The electrostatic interaction allows for easy fabrication of the particles and facilitates release of the active agent. Layer-by-layer deposition techniques may be used to coat the particles. For example, particles may be suspended in a solution containing the coating material, which then simply adsorbs onto the surface of the particles. The coating is not a thick or tight layer but rather allows the active agent to diffuse from the polymer core into the bloodstream. In addition, where enzymatic action is needed to decompose the core, the coating allows enzymes to diffuse from the blood into the particle. Even though the coating can remain intact as the active agent is released, it is itself susceptible to decomposition, and the particle can be fully metabolized.
In addition to electrostatic interactions, other non-covalent interactions may also be used to immobilize a coating. Exemplary non-covalent interactions include but are not limited to the following:
1) Affinity Interactions: For example, biotin may be attached to the surface of the controlled release polymer core and streptavidin may be attached to the coating material; or conversely, biotin may be attached to the coating material and the streptavidin may be attached to the surface of the controlled release polymer core. The biotin group and streptavidin are typically attached to the controlled release polymer system or to the coating via a linker, such as an alkylene linker or a polyether linker. Biotin and streptavidin bind via affinity interactions, thereby retaining the coating on the controlled release polymer core.
2) Metal Coordination: For example, a polyhistidine may be attached to the coating material, and a nitrilotriacetic acid can be attached to the surface of the controlled release polymer core. A metal, such as Ni⁺², will chelate the polyhistidine and the nitrilotriacetic acid, thereby binding the coating to the controlled release polymer core.
3) Physical Adsorption: For example, a hydrophobic tail, such as polymethacrylate or an alkyl group having at least about 10 carbons, may be attached to the coating material. The hydrophobic tail will adsorb onto the surface of a hydrophobic controlled release polymer, such as a polyorthoester, polysebacic anhydride, unmodified poly(lactic acid), or polycaprolactone, thereby binding the coating to the controlled release polymer core.
4) Host-Guest Interactions: For example, a macrocyclic host, such as cucurbituril or cyclodextrin, may be attached to the controlled release polymer or the surface of the controlled release polymer core and a guest group, such as an alkyl group, a polyethylene glycol, or a diaminoalkyl group, may be attached to the coating material; or conversely, the host group may be attached to the coating material and the guest group may be included in the controlled release polymer core. In one embodiment, the host and/or the guest molecule may be attached to the coating material or the controlled release polymer system via a linker, such as an alkylene linker or a polyether linker.
5) Hydrogen Bonding Interactions: For example, an oligonucleotide having a particular sequence may be attached to the surface of the controlled release polymer core, and an essentially complementary sequence may be attached to the coating material. The coating material will then bind to the controlled release polymer core via complementary base pairing with the oligonucleotide attached to the controlled release polymer system. Two oligonucleotides are essentially complimentary if about 80% of the nucleic acid bases on one oligonucleotide form hydrogen bonds via an oligonucleotide base pairing system, such as Watson-Crick base pairing, reverse Watson-Crick base pairing, Hoogsten base pairing, etc., with a base on the second oligonucleotide. Typically, it is desirable for an oligonucleotide sequence attached to the controlled release polymer system to form at least about 6 complementary base pairs with a complementary oligonucleotide attached to the nucleic acid ligand. For example, a poly(cytosine) tag may be attached to the controlled release polymer core and a poly(guanine) tag may be attached to the coating material. Indeed, it is not necessary to only surface treat the controlled release polymer; the entire polymer may be so modified. Some of the poly-C tags will end up on the surface of the core, and others will remain in the interior portions of the particle. In another embodiment, sugars may be used as a mucoadhesive coating. The hydroxyl groups on sugars such as glucose and galactose will hydrogen bond with polar moieties on polymers such as poly(vinyl alcohol). Sugar dimers or oligomers may be used as well.
The core and the coating may also be linked via covalent interactions. For example, PLGA may be modified with a carboxylate group and employed as a core material. Chitosan or another aminated coating material can be coupled to the core using a coupling reagent such as EDC or DCC. Alternatively, PLGA may be modified to have an activated NHS ester which can then be reacted with an amine group on the coating material. Either coating or core materials may be modified to include reactive groups such as hydroxyl, amine, carboxyl, maleimide, thiol, NHS ester, azide, or alkyne. Standard coupling reactions may then be used to couple the modified material to a second material having a complementary group (e.g., a carboxyl modified core coupled to an aminated coating material).

Administration of Inventive Compositions

Once the inventive particles have been prepared, they may be combined with pharmaceutical acceptable carriers to form a pharmaceutical composition. While the composition may be injectable or administrable as a suppository, it is preferable that the composition be orally administrable, either through ingestion or as an inhalant. To this end, the particles produced using the techniques described herein may be sufficiently small to traverse the intestinal mucosa or the alveolar wall. Enhanced uptake may be achieved for larger particles by the use of mucoadhesive coatings, as described herein. The size of the particle may be optimized for stability and increased uptake. One skilled in the art will recognize that the optimum particle size may vary depending on the nature of the drug being delivered. The studies described below may be used to determine the optimal particle size.
As used herein, the term “pharmaceutically acceptable carrier” means a non-toxic, inert solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type. Remington's Pharmaceutical Sciences Ed. by Gennaro, Mack Publishing, Easton, Pa., 1995, discloses various carriers used in formulating pharmaceutical compositions and known techniques for the preparation thereof. Some examples of materials which can serve as pharmaceutically acceptable carriers include, but are not limited to, sugars such as lactose, glucose, and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium carboxymethyl cellulose, ethyl cellulose, and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients such as cocoa butter and suppository waxes; oils such as peanut oil, cottonseed oil; safflower oil; sesame oil; olive oil; corn oil and soybean oil; glycols such as propylene glycol; esters such as ethyl oleate and ethyl laurate; agar; detergents such as TWEEN™ 80; buffering agents such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol; and phosphate buffer solutions, as well as other non-toxic compatible lubricants such as sodium lauryl sulfate and magnesium stearate. Coloring agents, releasing agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and/or antioxidants can also be present in the composition, according to the judgment of the formulator.
The pharmaceutical compositions of the invention can be administered to a patient by any means known in the art including oral and parenteral routes. The term “patient”, as used herein, refers to humans as well as non-humans, including, for example, mammals, birds, reptiles, amphibians, and fish. Preferably, the non-humans are mammals (e.g., a rodent, a mouse, a rat, a rabbit, a monkey, a dog, a cat, a primate, or a pig). Non-edible compositions may be administered by injection (e.g., intravenous, subcutaneous or intramuscular, intraperitoneal injection), rectally, vaginally, topically (as by powders, creams, ointments, or drops), or by inhalation (as by sprays).
Powders and sprays can contain, in addition to the inventive particles of this invention, excipients such as lactose, talc, silicic acid, aluminum hydroxide, calcium silicates, and polyamide powder, or mixtures thereof. Sprays can additionally contain customary propellants such as chlorofluorohydrocarbons.
Pharmaceutical compositions for oral administration can be liquid or solid. Liquid dosage forms suitable for oral administration of inventive particles include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups, and elixirs. In addition to an encapsulated or unencapsulated particle, the liquid dosage forms may contain inert diluents commonly used in the art such as, for example, water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor, and sesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof. Besides inert diluents, the oral compositions can also include adjuvants, wetting agents, emulsifying and suspending agents, sweetening, flavoring, and perfuming agents. As used herein, the term “adjuvant” refers to any compound which is a nonspecific modulator of the immune response. In certain preferred embodiments, the adjuvant stimulates the immune response. Any adjuvant may be used in accordance with the present invention. A large number of adjuvant compounds is known in the art (Allison Dev. Biol. Stand. 92:3-11, 1998; Unkeless et al. Annu. Rev. Immunol. 6:251-281, 1998; and Phillips et al. Vaccine 10:151-158, 1992).
Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules. In such solid dosage forms, the encapsulated or unencapsulated particle is mixed with at least one inert, pharmaceutically acceptable excipient or carrier such as sodium citrate or dicalcium phosphate and/or (a) fillers or extenders such as starches, lactose, sucrose, glucose, mannitol, and silicic acid, (b) binders such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidinone, sucrose, and acacia, (c) humectants such as glycerol, (d) disintegrating agents such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate, (e) solution retarding agents such as paraffin, (f) absorption accelerators such as quaternary ammonium compounds, (g) wetting agents such as, for example, cetyl alcohol and glycerol monostearate, (h) absorbents such as kaolin and bentonite clay, and (i) lubricants such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof. In the case of capsules, tablets, and pills, the dosage form may also comprise buffering agents.
Solid compositions of a similar type may also be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugar as well as high molecular weight polyethylene glycols and the like. The solid dosage forms of tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells such as enteric coatings and other coatings well known in the pharmaceutical formulating art.
It will be appreciated that the exact dosage of the inventive particle is chosen by the individual physician in view of the patient to be treated. In general, dosage and administration are adjusted to provide an effective amount of the desired active agent to the patient being treated. As used herein, the “effective amount” of a substance refers to the amount necessary to elicit the desired biological response. As will be appreciated by those of ordinary skill in the art, the effective amount of encapsulated active agent may vary depending on such factors as the desired biological endpoint, the active agent to be delivered, the target tissue, the route of administration, etc. For example, the effective amount of inventive particles containing an anti-cancer drug might be the amount that results in a reduction in tumor size by a desired amount over a desired period of time. Additional factors which may be taken into account include the severity of the disease state; age, weight and gender of the patient being treated; diet, time and frequency of administration; drug combinations; reaction sensitivities; and tolerance/response to therapy.
The particles of the invention are preferably compounded with a carrier in dosage unit form for ease of administration and uniformity of dosage. The expression “dosage unit form” as used herein refers to a physically discrete unit of conjugate appropriate for the patient to be treated. It will be understood, however, that the total daily usage of the compositions of the present invention will be decided by the attending physician within the scope of sound medical judgment. For any particle composition, the therapeutically effective dose can be estimated initially either in cell culture assays or in animal models, usually mice, rabbits, dogs, or pigs. The animal model is also used to achieve a desirable concentration range and route of administration. Such information can then be used to determine useful doses and routes for administration in humans. Therapeutic efficacy and toxicity of particle materials and the drugs delivered thereby can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., ED₅₀(the dose is therapeutically effective in 50% of the population) and LD₅₀(the dose is lethal to 50% of the population). The dose ratio of toxic to therapeutic effects is the therapeutic index, and it can be expressed as the ratio, LD₅₀/ED₅₀. Pharmaceutical compositions which exhibit large therapeutic indices are preferred. The data obtained from cell culture assays and animal studies is used in formulating a range of dosage for human use.

EXAMPLES

Example 1: In Vitro and In Vivo Evaluation of Insulin Release from Nano- or Microspheres with or without Bioadhesive Polymer Coats

Encapsulation of Insulin in Nano- or Microspheres
Poly(D,L-lactic acid) containing carboxylic end group (PLA-COOH, inherent viscosity=0.15-0.25 corresponding to 11 k-25 k in molecular weight) was purchased from Birmingham Polymers, Inc. (Birmingham, Ala.). OH-PEG₃₄₀₀-COOH was custom synthesized by Nektar Therapeutics (San Carlos, Calif., USA). ¹²⁵I-labeled insulin was purchased from Amersham Bioscience (Piscataway, N.J., USA). Chitosan (minimum 85% deacetylated), poly(vinylalcohol) (PVA), and D(+)-trehalose were also obtained from Sigma.
Poly(D,L-lactic acid)-block-poly(ethylene glycol)-COOH (PLA-PEG₃₄₀₀-COOH) was synthesized by ring opening polymerization with minor modifications in anhydrous toluene using stannous octoate as catalyst. D,L-Lactide (1.6 g, 11.1 mmol) and COOH-PEG₃₄₀₀-OH (290 mg, 0.085 mmol) in anhydrous toluene (10 mL) containing anhydrous Na₂SO₄(200 mg) were heated to reflux temperature at 120° C., after which the polymerization was initiated by adding tin(II) 2-ethylhexanoate (20 mg, 0.049 mmol). After stirring for 6 h with reflux, the reaction mixture was cooled to room temperature. To this solution was added cold water (10 mL), and the resulting suspension was stirred vigorously at room temperature for 30 min to hydrolyze unreacted lactide monomers. The resulting mixture was transferred to a separation funnel containing CHCl₃(50 mL) and water (30 mL). After layer separation, the organic layer was collected, dried using anhydrous MgSO₄, filtered, and concentrated under reduced vacuum. Hexane was then added to the concentrated solution to precipitate the polymer product. Pure PLA-PEG₃₄₀₀-COOH was collected as a white solid. PLA-PEG₃₄₀₀-COOH: ¹H-NMR (400 MHz), δ=5.28-5.11 (br, —OC—CH(CH₃)O— in PLA), 3.62 (s, —CH₂CH₂O— in PEG), 1.57-1.45 (br, —OC—CHCH₃O— in PLA); molecular weight (GPC): M_n=10500 with M_w/M_n=1.54 relative to monodisperse polystyrene standards. ¹H NMR (400 MHz) spectra were recorded on a Bruker instrument (Avance DPX 400). Aqueous phase GPC was performed by American Polymer Standards (Mentor, Ohio) using Ultrahydrogels L and 120 A columns in series (Waters Corporation, Milford, Mass.). Water (1% acetic acid, 0.3 M NaCl) was used as the eluent at a flow rate of 1.0 mL/min. Data were collected using a Knauer differential refractometer and processed using an IBM/PC GPC-PRO 3.13 software package (Viscotek Corporation, Houston, Tex.).
Drug encapsulated nanoparticles were prepared using the water-in-oil-in-water (W/O/W) solvent evaporation procedure (double emulsion method) employed elsewhere. In brief, 50 μl of the ¹²⁵I-labeled Insulin solution (1 mg/mL in aqueous 2 wt % trehalose) was emulsified in a 1 mL solution of the polymer (PLA-COOH or PLA-PEG-COOH) (50 mg) in dichloromethane using a probe sonicator (10 W for 15-20 s). To this emulsion was then added 3 mL of aqueous PVA (1% w/v), and the mixture was sonicated again for 20 s (10 W) using the same probe sonicator. The resulting emulsion was poured into 50 mL of aqueous PVA (0.3% w/v) with gentle stirring, after which organic solvent was rapidly removed using rotary evaporator. Finally, the ¹²⁵I-Insulin encapsulated nanoparticles were isolated by centrifugation at 10,000 rpm for 10 min, washed 2 times with water, and preserved at −15° C. as emulsion form in aqueous trehalose (2% w/v, 2 mL).
Chitosan-coated nanoparticles were prepared as follows. The ¹²⁵I-Insulin encapsulated nanoparticles (1 mL, ca. 10 mg) were added dropwise to chitosan solution (10 mL, 0.2 wt % in distilled water, pH 5.5) with gentle stirring. After 10 min, the resulting suspension was then centrifuged at 10,000 rpm for 10 min. The remained nanoparticles were washed with aqueous trehalose (2% w/v, 20 mL) and finally suspended in aqueous trehalose (2% w/v, 2 mL).
Characterization of Particles
Nanoparticles encapsulating ¹²⁵I-labeled were prepared by the procedure above, centrifuged, and then the radioactivity in the supernatant was measured by liquid scintillation analyzer (Packard Instrument Company, Downers Grove, Ill.). The encapsulation efficiency was calculated by the difference between the total amount of radioactivity in the initial solution and the remained amount in the supernatant.
The nanoparticles before and after chitosan coating were characterized using several standard analytical means. The size of the particles and zeta-potential (surface charge) were measured by Quasi-elastic laser light scattering (QELS) using a ZetaPALS dynamic light scattering detector (Brookhaven Instruments Corporation, 15 mW laser, incident beam=676 nm). Samples (0.1 mL, ca. 1 mg) were diluted with 3 mL of distilled water. Particle sizes were measured at 25° C. Correlation functions were collected at a scattering angle of 90°, and particle sizes were calculated using the MAS option of the company's particle sizing software (version 2.30) under the viscosity and refractive index of pure water at 25° C. Particle sizes expressed as effective diameters assuming a log-normal distribution. Three measurements were made on each sample and results are reported as mean diameters. Electrophoretic mobilities were measured at 25° C. using BIC PALS zeta potential analysis software, and zeta potentials were calculated using the Smoluchowsky model. Surface morphology and size were characterized by high-resolution scanning electron microscopy (JEOL 6320FV). All samples were coated with 75 Å Au/Pd prior to analysis. Atom composition of the nanoparticles was analyzed using a Kratos AXIS Ultra Imaging X-ray Photoelectron Spectrometer with a monochromatized Al K X-ray source and a 160 mm concentric hemispherical energy analyzer for acquisition of spectra and scanned images, lateral resolution down to 20 μm. The spot size was 300 by 700 μm.
Table 1 summarizes the size, zeta potential, and drug encapsulation efficiency of the nanoparticles. As a model protein drug, ¹²⁵Iodine-labeled insulin was encapsulated by PLA-COOH and PLA-PEG-COOH polymer system with efficiency of 65% and 71%, respectively. Both polymer particles showed large negative zeta potentials, indicating the presence of the negatively charged carboxylic acid group on the particle surface. In particular, a much larger negative charge was developed on the surface of PEGylated particles compared to PLA particles. This may be attributed to the presence of the hydrophilic PEG block interposed between PLA and COOH. Upon exposure to aqueous media, COOH is sequestered at the particle surface. Chitosan coating was carried out by simply adding the negatively charged particles to chitosan solution at pH 5.5. Highly positive zeta potential values are observed after chitosan coating, suggesting successful coating of the cationic chitosan onto the negatively charged nanoparticles. As expected, there were slight increases in particle sizes after chitosan coating.

TABLE 1

Particle size, zeta potential, and drug encapsulation efficiency
of PLA-COOH and PLA-PEG-COOH based nanoparticles
encapsulating insulin

	Mean Size	Zeta Potential	Encapsulation
Polymer particles	(nm)^a	(mV)^a	efficiency (%)^a

PLA-COOH	275 ± 16	−35 ± 3	65 ± 7
PLA-COOH/Chitosan	310 ± 19	+55 ± 7
PLA-PEG-COOH	249 ± 12	−50 ± 3	71 ± 8
PLA-PEG-COOH/	265 ± 22	+59 ± 5
Chitosan

^aMean ± SD (n = 3).

The scanning electron microscopic images of the particles are shown in FIG. 1. A majority of the PLA-COOH and PLA-PEG-COOH nanoparticles are approximately 200 to 300 nm, and there was no distinct discrepancy before and after chitosan coating, in agreement with the quasi-elastic laser scattering (QELS) data.
The presence of a chitosan layer on the particle surface was further confirmed by X-ray photoelectron spectroscopy (XPS). We compared high resolution carbon (1 s) intensity. PLA contains three different types of carbons, namely, —C(═O)O carbonyl, C—O ether, and C—C carbons, whereas the PEG chain contains only C—O ether carbons. Very similarly, chitosan is also composed of mostly C—O ether carbon. Therefore, it is expected that after chitosan coating, the ratio of C—O to —C(═O)O carbons should increase. As shown in Table 2, the value increased by approximately 35% for PLA particles and 20% for PEGylated particles, respectively. These data also indicate that chitosan is coated onto the particle surface.

TABLE 2

High resolution XPS C(1s) composition of nanoparticles^a

Composition (%)^b

Nanoparticles	C—C	C(═O)O	C—O	C—O/C(═O)O

PLA-COOH	31.1 ± 0.7	32.8 ± 0.3	36.1 ± 0.5	1.10
PLA-COOH^c	30.5 ± 0.4	28.0 ± 0.5	41.5 ± 0.2	1.48
PLA-PEG-COOH	42.2 ± 0.8	26.5 ± 0.6	31.2 ± 0.7	1.18
PLA-PEG-	40.5 ± 0.4	24.6 ± 0.3	35.0 ± 0.2	1.42
COOH^c

^a55 take-off angle was taken from surface normal.
^bMean ± SD.
^cChitosan-coated equivalent.

Evaluation of Particles in Simulated Digestive Fluid
Simulated gastric fluid (pH 1.2, pepsin 0.32% w/v, sodium chloride 0.2% w/v) and intestinal fluid (pH 7.5, pancreatin 1% w/v, monobasic potassium phosphate 0.68% w/v) were prepared by referring to US Pharmacopoeia (23^rdedition, 1995). Pepsin from porcine stomach mucosa and pancreatin from porcine pancreases were purchased from Sigma (St Louis, USA). Chitosan-coated and uncoated nanoparticles were incubated in each digestive solution for 4 h. Degradation of PLA was determined by measuring lactate concentration in the suspension using a lactate detection kit. Briefly, each nanoparticle (5 mg) encapsulating FITC-labeled insulin (as a model protein) was incubated at 37° C. in simulated digestive fluid (5 mL) with gentle stirring using a magnetic bar. Aliquots (33 μL) were removed at appropriate time intervals and mixed with 967 μL of lactate detection kit (0.5 mL lactate dehydrogenase, 2 vials nicotinamide adenine dinucleotide, 12 mL glycine buffer). After incubation at 37° C. for 15 min, the absorbance of the solution was measured at 340 nm.
Particle stability was determined by measuring lactate concentration in solution after neutralization using a lactate detection kit. As shown in Table 3, in gastric fluid, 3% and 2% of the PLA in PLA-COOH and PLA-PEG-COOH particles, respectively, degraded. In intestinal fluid, the degradation percentage was ca. 15% and 10%, respectively. As expected, the presence of PEG increased stability as compared to simple PLA, presumably due to the enzyme repelling properties of PEG. The chitosan coating rendered the corresponding nanoparticles more stable than the uncoated equivalents in intestinal fluid (15% versus 9% degradation for PLA-COOH and 10% versus 6% degradation for PLA-PEG-COOH nanoparticles, respectively). In addition, when the chitosan-coated PLA-PEG-COOH nanoparticles were collected and analyzed after 4 h incubation in gastric fluid, the size and zeta potential of collected nanoparticles were unchanged, suggesting the chitosan coating layer remained stable on the particle surface (data not shown). Consequently, coexistence of PEG and chitosan coating layers largely increased particle stability over PLA nanoparticles in digestive fluids.

TABLE 3

Stability of the nanoparticles in simulated gastric and intestinal fluids

PLA converted to lactate (%)^a

Nanoparticles	Gastric fluid (4 h)	Intestinal fluid (4 h)

PLA-COOH	3 ± 0.9	15 ± 1.3
PLA-COOH ^b	2 ± 0.5	9 ± 2.2
PLA-PEG-COOH	2 ± 0.3	10 ± 1.5
PLA-PEG-COOH ^b	2 ± 1.1	6 ± 0.8

^aMean ± SD.
^bChitosan-coated equivalent.

In Vitro Model: Determination of Uptake Efficiency of Chitosan-Coated Controlled Release Polymer Particles Across Human Intestinal Epithelial Cells (Caco-2)
All manipulations involving live cells or sterile materials were performed in a laminar flow hood using standard sterile technique. The Caco-2 cell line was purchased from the American Type Culture Collection (Manassas, Va.) and grown at 37° C., 5% CO₂in Dulbecco's modified Eagle's medium (DMEM), 90%; fetal bovine serum, 10%; penicillin, 100 units/mL; streptomycin, 100 μg/mL; apo-transferrin, 10 μg/mL. Costar transwells (12 well/plate) were obtained from Corning, Inc. (Acton, Mass.).
The Caco-2 cells were seeded at 1.7×10⁵cells/mL in 12 well/plate transwells (Costar, pore size of 3 μm) and grown for 12-24 days. Costar transwells had a membrane insert size of 12 mm and a pore size of 3 μm, with an apical chamber volume of 0.75 mL and a basal chamber volume of 2 mL. Cells generally reached confluence within 10 days. Transepithelial resistance measurements were made of monolayers and only cultures with greater than 250 ohms/cm²were used for experiments representing a “tight monolayer” with closed gap junctions. 100 μL of each nanoparticle from stock solution (ca.10 mg/mL in aqueous 2% w/v trehalose) was mixed with 400 μL of cell culture medium and then, the mixture was loaded to each apical chamber. After 12 h, the radioactivity in the basal chamber was measured using a liquid scintillation analyzer to measure the transport efficiency of the particles across the Caco-2 monolayers. The transport efficiency was denoted as % total radioactivity obtained in the basal chamber. The results are summarized in FIG. 2.
First, in terms of chitosan coating effect, both non-PEGylated and PEGylated nanoparticles showed approximately 40% increased transport after chitosan coating. Enhanced transport by chitosan coating may be attributed to either increased interaction of positively charged chitosan with partially negatively charged epithelial cell layers or chitosan-facilitated paracellular transport. Although the mechanism by which chitosan can open tight junctions is not clearly demonstrated to date, it is currently accepted that it opens the junction reversibly so as to facilitate paracellular transport of macromolecules. Secondly, PEGylated polymer particles exhibited much higher (at least 60%) transport than non-PEGylated systems with and without chitosan coating. Therefore, the chitosan-coated PEGylated nanoparticle appears to have the most satisfactory transport profile in this experimental model.
In Vivo: Absorption and Biodistribution of Nanoparticles in Mice
Nanoparticles (0.2 mL) encapsulating ¹²⁵I-labeled insulin were fed to mice (n=8) by a gavage method. Male BALA/c mice (10 weeks old, 22-25 g weight) were obtained from Taconic (Germantown, N.Y.) and allowed access to food and water. At 1 h after oral administration, half of the mice (n=4) were sacrificed, then samples of blood (0.2 mL) were taken by cardiac puncture. The remaining mice (n=4) were sacrificed at 6 h post oral administration and samples of blood were taken. Some organs and tissues were harvested by necropsy. The radioactivity in blood or in tissues was measured to calculate absorption yield into the body.
The results are summarized in FIG. 3. The absorption yield of insulin into blood was denoted as % original dose. The chitosan-coated particles exhibited approximately 80% increased insulin-absorption at 1 h and 40% increased absorption when measured at 6 h with respect to their uncoated equivalent. Much less absorption was observed for microparticles than for nanoparticles, but they both still exhibited enhanced insulin uptake after chitosan coating. Chitosan coating of nanoparticles resulted in at least 2 times higher absorption of insulin into the blood than corresponding microparticles. The absorption yield of chitosan-coated nanoparticles (14%) indicates an efficiency several times higher than reported literature values of 2-3%.

Example 2: In Vivo Absorption of ¹²⁵I-Insulin and Humulin R

General
Human insulin (Humulin R, 100 U/mL and 500 U/mL, 100 unit=3.5 mg Humulin R) was purchased from www.drugstore.com and used as the stock solution. Bovine zinc insulin was purchased from Sigma. A number of different polymers used in these studies were obtained from Absorbable Polymers International (Pelham, Ala.): low molecular-weight (LMW) poly(DL-lactic acid) with acid terminal groups (LMW PLA-COOH, inherent viscosity (IV) 0.20 dL/g,), high molecular-weight (HMW) poly(DL-lactic acid) with acid terminal groups (HMW PLA-COOH, IV 0.45 dL/g), LMW 50/50 poly(DL-lactide-co-glycolide) with acid terminal groups (LMW PLGA-COOH, IV 0.18 dL/g), HMW 50/50 poly(DL-lactide-co-glycolide) with acid terminal groups (HMW PLGA-COOH, IV 0.76 dL/g). NH₂-PEG₅₀₀₀-COOH and HO-PEG₅₀₀₀-COOH were purchased from Nektar Therapeutics. ¹²⁵I-labeled insulin was purchased from Amersham Bioscience (Piscataway, N.J., USA). Chemicals purchased from Sigma/Aldrich Chemical Co include Chitosan (minimum 85% deacetylated), poly(vinylalcohol) (PVA), and D(+)-trehalose tin(II) 2-ethylhexanoate, D,L-lactide, toluene (99.8%, anhydrous), and lectin from Bandeiraea Simpliciforia BS-1.

Synthesis of PLA-PEG-COOH Via Ring-Opening Polymerization

PLA-PEG-COOH was synthesized through ring opening polymerization of DL-lactide in anhydrous toluene using stannous octoate as catalyst. DL-Lactide was recrystalized with ethyl acetate prior to polymerization (using approximately 250 mL ethyl acetate to recrystallize 50 g DL-lactide). DL-lactide (15 mmol) and COOH-PEG₃₄₀₀-OH (0.1 mmol) in anhydrous toluene (10 mL) were heated to 120° C. under N₂. Tin(II) 2-ethylhexanoate (30 mg) was added to the solution to initiate polymerization. After stirring for 6-8 h under N₂at 120° C., the reaction mixture was cooled to room temperature and poured to cold methanol (60 mL). The precipitate was washed with methanol and dried under vacuum (yield 62%). ¹H NMR (400 MHz) δ=5.20 (m, —OC—CH(CH₃)O—), 3.55 (s, —CH₂CH₂O—), 1.57-1.45 (br, —OC—CHCH₃O—).

Synthesis of PLGA-PEG-COOH (or PLA-PEG-COOH) Via Conjugation Methods

HMW DL-PLGA-COOH (0.99 g) was dissolved in anhydrous methylene chloride (5 mL). N-Hydroxysuccinimide (NHS) (23 mg) and 1-Ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) (42 mg) were added to the solution. After 3 h solution was poured into 25 mL cold MeOH. The precipitate was washed with 3×25 mL MeOH and dried under vacuum (yield 100%). The white solid (335 mg) obtained was re-dissolved in chloroform (3.35 mL). A chloroform solution of NH₂-PEG₅₀₀₀-COOH (32.5 mg in 320 chloroform) was added dropwisely to the activated PLGA solution. Diisopropylethylamine (DIEA, 3 eq., 2.52 mg) was added the mixtures. The solution was stirred under N₂overnight. The resulting PLGA-PEG-COOH was precipitated with cold MeOH and dried under vacuum (yield 95%). ¹H NMR (400 MHz) δ=5.18 (m, —OC—CH(CH₃)O—), 4.79 (m, —OC—CH₂O—), 3.61 (s, —CH₂CH₂O—), 3.41 (s, —CH₂CH₂O—), 1.56 (br, —OC—CHCH₃O—).

Preparation of ¹²⁵-Insulin-Encapsulated Nanoparticles (NPs)

Drug encapsulated NPs were prepared using the water-in-oil-in-water (W/O/W) solvent evaporation procedure (double emulsion method). A 50 μl solution of the ¹²⁵I-Insulin solution (1-10 μCi/mL reconstituted in pH 7.4 PBS) was emulsified in a 1 mL dichloromethane solution of 50 mg corresponding polymer (PLA-COOH, PLA-PEG-COOH) in using a probe sonicator (Sonic & Materials Inc, Danbury, Conn., USA) at 10 W for 30 s. To this emulsion was then added 3 mL of aqueous PVA (MW=30-70 kDa, 1% w/v) and the mixture was sonicated for 30 s using the same probe sonicator at 10 W. The resulting emulsion was immediately poured into 50 mL of aqueous PVA (MW=30-70 kDa, 0.3% w/v) with gentle stirring. Organic solvent was either rapidly removed using rotary evaporator or by stirring at room temperature for 2.5 h. The NPs were isolated by centrifugation at 10000 rpm for 10 min at 10° C., washed once with double-distilled water, and used for study immediately. The yield of NPs was between 20 and 30%.

Preparation of ¹²⁵-Insulin-Encapsulated Microparticles (MPs)

Drug encapsulated MPs were prepared using the water-in-oil-in-water (W/O/W) solvent evaporation procedure (double emulsion method). 50 μl of the ¹²⁵I-Insulin solution (1-10 μCi/mL in pH 7.4 PBS) was emulsified in a 1 mL dichloromethane solution of 50 mg corresponding polymer (PLA-COOH, PLA-PEG-COOH) using a probe sonicator at 10 W for 30 s. The first emulsion was transferred to 50 mL of aqueous PVA (MW=30-70 kDa, 1% w/v) and homogenized at 8000 rpm for 1 minute. The resulting emulsion was immediately poured into 200 mL of aqueous PVA solution (MW=30-70 kDa, 0.3% w/v) with gentle stirring. Organic solvent was removed using rotary evaporator or by stirring at room temperature for 2.5 h. The MPs were isolated by centrifugation at 10000 rpm for 10 min at 10° C., washed once with double-distilled water, and lyophilized. The yield of MPs was between 60-80%.

Preparation of Humulin R-Encapsulated NPs and MPs

The procedures for making Humulin R-encapsulated NPs and MPs are same as that for making ¹²⁵I-insulin-encapsulated NPs and MPs (see above) except that 50 μL of 500 U/mL Humulin R was used instead of ¹²⁵I-insulin PBS solution.
Chitosan Coating of NP and MPs
To a suspension of NPs (or MPs) (15-30 mg in 1 mL water) was added a chitosan solution (20 mL, 0.2 wt % in distilled water, pH 5). After the suspension is allowed to stand for 10 min, the resulting suspension was centrifuged at 10000 rpm for 10 min. The NP (or MPs) were washed with 2% trehalose solution, centrifuged, and stored at −20° C. The chitosan-coated NP and MPs prepared for in vivo glucose reduction were washed with water instead of trehalose solution and used immediately.
NP Modified with Lectin
Lectin from Bandeiraea simplicifolia (Sigma) was reconstituted with DI water. LMW PLGA-COOH NP (20 mg) was activated with EDC/NHS and then reacted with lectin (0.2 mg) in the presence of excess DIEA (1 mg). The suspension was stirred for 4 h at room temperature. The resulting NPs were washed with water, centrifuged twice, and stored in 2 mL water at −15° C.
Characterization of the NPs and MPs
The size of the NPs and the zeta-potential of NPs and MPs were measured by Quasi-elastic laser light scattering (QELS) using a ZetaPALS dynamic light scattering detector (Brookhaven Instruments Corporation, 15 mW laser, incident beam=676 nm). Particle sizes were measured at room temperature at a concentration of 0.5-1 mg/mL water. Three to five measurements were made on each sample. Results are reported as mean diameters. Electrophoretic mobilities were measured at 25° C. using BIC PALS zeta potential analysis software, and zeta potentials were calculated using the Smoluchowsky model. Surface morphology and size were characterized by scanning electron microscopy.
Determination of Drug Loading
The loading of ¹²⁵I-insulin in NPs and MPs was determined by analyzing the solution of known amount of NPs or MPs (2 mg NPs or MPs dissolved in 1 mL acetonitrile/water with 5 mL added Hionic-Fluro cocktail) using a Liquid Scintillation Analyzer (Packard Instrument Company, Downers grove, IL). The loading of Humulin R was determined by measuring the actual amount of Humulin R using a BCA protein assay (Pierce Chemical Co., Rockford, Ill.). Briefly, aliquots containing known amounts of particles (5-10 mg) dissolved in 2 ml of CH₂Cl₂were extracted with 2 ml buffer (1×phosphate-buffered saline (PBS) and 0.05% sodium dodecyl sulfate (SDS)). Humulin R in buffer solution was quantified using BCA assay (Pierce) in triplicate.
In-Vivo Absorption of ¹²⁵I-Insulin Encapsulated PLA-Based NPs and MPs
The ¹²⁵I-labelled insulin was encapsulated into NPs/MPs using carboxylate terminated poly(lactic acid) (PLA-COOH) or poly(lactic acid)-co-poly(ethylene glycol) (PLA-PEG-COOH) by the double emulsion method. The surface of the resulting NPs/MPs were negatively charged, which facilitates use of a positively charged mucoadhesive material (e.g., chitosan) coating. NPs were typically in a range of 300-400 nm, while MPs were in a range of 1-10 μm. Details for the synthesis of PLA-PEG-COOH and for the preparation and characterization NPs/MPs are given above.
¹²⁵I-Insulin encapsulated PLA or PLA-PEG NPs and MPs (Table 3) were administered to mice (n=4) orally by gavage. Each animal received 0.2 mL of the NP or MP suspension (approximately 0.2-0.5 KO. Mice were euthanized at 6 h by CO₂inhalation. Blood (200 μL) from each mouse was collected immediately in a glass scintillation vial by cardiac puncture. The blood was treated with 0.4 mL Solvable for 1 h at 55° C. Sample color turned into green. EDTA-di-sodium salt solution (0.04 mL) was added, followed by dropwise addition of 30% hydrogen peroxide (0.15 mL). The solution was allowed to stand for 15 to 30 minutes at room temperature to complete the reaction. The vial was incubated again at 55-60° C. for one hour. The color changed from green to pale yellow. After solution was cooled to room temperature for about 1 h, Hionic-Fluor (5 mL) was added to the mixture. The solution was then analyzed on a Liquid Scintillation Counter (Packard, Ill.).

TABLE 3

Nanoparticle and Microparticle Characterization

¹²⁵I-Insulin Encapsulated Nanoparticles	Particle size
(NP) and Microparticles (MP)	(nm)	Zeta Potential

PLA-COOH NP	332 ± 2.5	−28.42 ± 2.21
PLA-PEG-COOH NP	375 ± 6.2	−37.49 ± 1.73
PLA-COOH NP/Chitosan	405 ± 18.6	53.06 ± 0.77
PLA-PEG-COOH NP/Chitosan	415 ± 13.8	57.33 ± 0.90
PLA-PEG-COOH MP	1-5 μm	−35.70 ± 1.07

NPs/MPs (Table 3) were administered to Balb/C mice (average 25 g, N=4) by oral gavage at 0.2-0.5 μCi per mouse. Mice blood (200 μL) was collected and de-colored 6 h after administration and analyzed by liquid scintillation counter (Tri-Carb, Packard). PEGylated NPs exhibited a slightly lower systemic absorption with respect to non-PEGylated NPs for both chitosan-coated (4.79% for PLA-COOH NP and 4.41% for PLA-PEG-COOH NP) and uncoated systems (4.02% for PLA-COOH NP and 3.83% for PLA-PEG-COOH NP) (FIG. 4A). The absorption of NPs coated with chitosan was enhanced with respect to equivalent NPs without chitosan. The absorption of PLA-PEG-COOH NPs was increased by 133% as compared to PLA-PEG-COOH MPs (FIG. 4B). This in-vivo absorption study showed that particle size significantly affects the absorption efficiency and smaller particles can be absorbed more efficiently.
Release of Insulin from PLA-PEG-COOH and PLGA-PEG-COOH Particles
In the previous set of experiments we measured the amount of radioactivity (¹²⁵I insulin encapsulated particles) in the plasma. However, we did not differentiate between released insulin and insulin that remained encapsulated. Since only the released insulin is therapeutically effective, it is desirable to evaluate the insulin release rate from particles prepared from various polymers. In this set of studies, we used particles generated from the PLA-PEG-COOH as before, in addition to particles generated from a poly(DL-lactide-co-glycolide)-PEG-COOH (PLGA-PEG-COOH) polymer system (the latter is expected to have a faster release kinetics). We studied the release of insulin from uncoated and chitosan-coated PLA-PEG-COOH or PLGA-PEG-COOH particles in PBS. 68% of insulin was released from uncoated PLGA-PEG-COOH NPs within 4 h, while about 42% of insulin was released from uncoated PLA-PEG-COOH NPs. The insulin release rate from particles with chitosan surface-coatings was reduced by 10-15% in both the PLA-PEG-COOH and PLGA-PEG-COOH systems.
Oral Efficacy of Humulin R Encapsulated PLA-PEG-COOH and PLGA-PEG-COOH Nanoparticles
The detected radioactivity in blood is a combined effect of the released (both active and denatured), encapsulated and decomposed insulins. It is desirable to study the availibility of the free, active insulin to evaluate the effectiveness of and to further improve the polymer particles for oral insulin delivery. We determined bioavailability by measuring glucose concentrations and by quantifying released insulin in blood. Plasma glucose levels were obtained by using the Ascensia Breeze Blood Glucose Monitoring System (Bayer) following the manufacturer's protocols. Since NPs show higher in-vivo absorption than MPs (FIG. 4B), we only focused on NPs in this study.
We chose Humulin R because:

- 1. Humulin R consists of zinc-insulin crystals dissolved in a clear fluid. Humulin R has nothing added to change the speed or length of its action. It takes effect rapidly and has a relatively short duration of activity (about 4 hours) as compared with other insulins. Prolonged hypoglycemia obtained from a sustained release system can be attributed to the effectiveness of drug delivery vehicle.
- 2. Humulin R in mouse serum can be differentiated from mouse insulin using an appropriate assay, and thus can be accurately quantified.

We first chose to study the response of glucose concentration to the orally administered, Humulin R-encapsulated polymer NPs. BalB/C mice, weighing 23-25 g, were fasted for 12-16 h. The initial glucose level of each mouse was measured. Four mice were assigned to a group such that the mean values of the glucose concentrations of each group were roughly equal. Various amounts of Humulin R NPs or MPs in 200 μL water were administrated orally using gavage needles. Control mice were administered with 200 μL water only. The glucose level of each mouse was monitored at scheduled times. In some experiments and at scheduled times, in addition to measuring blood glucose concentration, we collected blood samples (50 μL) in heparinized tubes.
We found that PLA-PEG NPs generally are not as efficacious as PLGA-PEG-COOH NPs for reducing blood glucose (FIG. 5). PLGA-PEG-COOH NPs administered at 100 U/mg reduced glucose levels by 48.7±12.9%, compared to 24.0±5.93% after administration of the same dose using PLA-PEG-COOH NPs.
Administration of PLGA-PEG-COOH and PLA-PEG-COOH NPs at 50 U/kg reduced serum glucose by 36.2±7.5% and 16.7±9.3%, respectively (FIG. 5). The lowest glucose levels were observed 4 hours after administration in all tested groups except for the 50 U/kg dosed PLGA-PEG-COOH NP group, in which the lowest glucose level occurred 6 hours after administration. To determine the bioavailability, we measured the glucose concentrations after tail-vein intravenous (IV) administration of Humulin R alone at 0.5 U/kg (25-50 μL total, n=4) (FIG. 6). A rapid decrease of glucose concentration was observed during the first hour, and the lowest glucose level was detected at t=1 hour with a decrease of 45.4±12.2%. The glucose concentration returned to the original level after 5 hours (FIG. 6). Based on the percent of glucose level deviation from the fasting blood glucose level for the challenged mice (see FIG. 6 for the Humulin IV administration group), we calculated the area under the curve (AUC) for the percent of the decreased blood glucose (%) vs time (hour) using the trapezoidal method for both IV and oral administration groups (Table 4). Glucose bioavailibility after the oral administration of Humulin encapsulated in PLA-PEG-COOH NPs was 0.34±0.43 and 0.58±0.28 for the 50 U/kg and 100 U/kg groups, respectively. Glucose bioavailibility after administration of PLGA-PEG-COOH NPs, however, was 1.86±0.86 and 2.53±0.62 in the 50 U/kg and 100 U/kg groups, respectively. Thus, the bioavailability of glucose delivered using of PLGA-PEG-COOH was 320%-440% higher than after delivery using PLA-PEG-COOH. Without being limited by any particular hypothesis, the increased bioavailability of insulin delivered with PLGA may be due to the accelerated drug release rate compared to PLA groups (Carino, et al., Nanosphere based oral insulin delivery. Journal of Controlled Release 2000, 65, 261-269).

TABLE 4

Determination of Bioavailibility of Orally Administered Humulin R
Encapsulated PLA-PEG-COOH and PLGA-PEG-COOH Nanoparticles in Mice

			Dose	Avg Weight	AUC^a	Bioavailibility
Gp	Route	Substrate	(U/kg)	of Mice (g)	(%)	(f)^b

1	IV	Humulin R	0.5	29.7	95.79 ± 50.03	—
2	oral	Humulin R-PLA-	50	27.5	76.45 ± 94.60	0.34 ± 0.43
		PEG-COOH NP
3	oral	Humulin R-PLA-	100	27	133.27 ± 64.58	0.58 ± 0.28
		PEG-COOH NP
4	oral	Humulin R-PLGA-	50	27.5	206.59 ± 94.98	1.86 ± 0.86
		PEG-COOH NP
5	oral	Humulin R-PLGA-	100	28	276.07 ± 68.59	2.53 ± 0.62
		PEG-COOH NP

^aCalculated based on trapezoidal treatment of the percent of decreased blood glucose (%) vs time (hour) for both IV and oral administration groups.
^bf = [AUC_{0-8 h oral}× (weight of mice/dose)_oral]/[AUC_{0-5 h IV}× (weight of mice/dose)_IV]

Besides determining bioavailability based on the blood glucose concentration, we also studied bioavailibility by directly measuring serum Humulin R using an ELISA insulin assay (Mercodic, ALPCO Diagnostics). This assay has very high specificity for the detection of Humulin R in mouse serum and shows no cross-reaction with mouse insulin. As shown in FIG. 7, the concentration of Humulin R measured in mouse plasma is linear over a range typically used for quantification of absorbed Humulin R via oral administration. Blood (approximately 20 μL) samples were collected into Startedt serum gel microtubes and allowed to clot for 30 minutes. The tubes were then centrifuged for 3 min at 10,000×g. Serum (5 μL) was transferred to a micro-well and diluted with 20 μL calibrator 0 of the Mercodia insulin ELISA kit (ALPCO Diagnostics, Inc). Enzyme conjugate (100 μL) was added to each well. The mixture was incubated at room temperature for 1 h on a plate shaker. The reaction medium was aspirated and washed with 5×350 μL Wash Buffer (provided in the kit). The TMB substrate (200 μL) of the kit was added to the well and incubated for 15 min at room temperature. After adding 50 μL Stop Solution, the UV absorbance was measured at 450 nm. Insulin concentration was determined based on a standard curve generated from known concentration of insulin standard.
When serum was administered at 2 U/kg via tail-vein intravenous injection, the serum concentration decreased drastically from 3203.21±143.65 mU/L to 124.18±30.90 mU/L at t=1 h and to 14.17±3.08 mU/L at t=2 h (FIG. 8). Less than 0.5% of administered insulin remained in blood 2 h after administration. The AUC of insulin concentration (mU/kg) vs time (hour) was 1175.89±125.51 mU·hr/L (Table 5).
Since the encapsulation of insulin during double emulsion may denature and thus deactivate the loaded insulin, the bioactivity of some Humulin R encapsulated MPs were tested using intraperitoneal administration before testing for oral activity. Balb/c mice were fasted overnight (about 12-16 h). Humulin R and Humulin R-magnetite-PLGA-MP were injected intraperitoneally to one and three mice, respectively. Blood (1 drop, roughly 3 μL) was collected from tail vein through puncture using 26G needle and analyzed with Ascensia Breeze Blood Glucose Monitoring System. The serum concentration of the intraperitoneally (IP) administered Humulin (4 U/kg) reached its highest level (541.85±46.92 mU/L) at t=30 minutes, and decreased to 36.32±6.92 mU/L at t=7 h (FIG. 8). The AUC of insulin concentration (mU/kg) vs time (hour) of the IP administered insulin was 692.29±272.70 mU·hr/L. The bioavailability of IP administered insulin was 29.46±11.60%.
After oral administration of insulin-PLGA-PEG-COOH NP, chitosan-coated, insulin-PLGA-PEG-COOH NP, and insulin-PLGA-PEG-COOH NP-Lectin bioconjugates, the highest insulin concentrations were 267.08±10.07 mU/kg (t=2 h), 188.04±46.75 mU/kg (t=4 h), and 391.87±68.43 mU/kg (t=2 h), respectively (FIG. 9). By t=20.5 h, serum insulin concentrations decreased to 15.21±7.97 mU/kg, 8.05±14.46 mU/kg, 39.48±37.76 mU/kg, respectively. The bioavailabilities of insulin-PLGA-PEG-COOH NP, chitosan-coated insulin-PLGA-PEG-COOH NP, and insulin-PLGA-PEG-COOH NP-Lectin bioconjugates were 1.22±0.38%, 1.49±0.53%, and 1.87±0.65%, respectively. Chitosan coating and lectin conjugation of PLGA-PEG-COOH NP enhanced the bioavailability by 22% and 53%, respectively. The bioavailability of the insulin-PLGA-PEG-COOH NP (1.22%) obtained by measuring insulin concentration was 54-107% lower than that determined by measuring glucose concentrations (compared to bioavailability of 1.86% and 2.53% in Table 4).

TABLE 5

Determination of Bioavailibility of Orally Administered Humulin R
Encapsulated PLGA-PEG-COOH Nanoparticles with Various Surface Modifications

			Dose	AUC^a	Bioavailibility
Gp	Route	Substrate	(U/kg)	(mU · hr/L)	(f)^b

6	IV	Humulin R		2	1175.89 ± 125.51	—
7	IP	Humulin R		4	692.29 ± 272.70	29.43 ± 11.60
8	oral	Humulin R-PLGA-PEG-	200	1435.63 ± 445.84	1.22 ± 0.38
		COOH NP
9	oral	Humulin R-PLGA-PEG-	200	1754.87 ± 629.15	1.49 ± 0.54
		COOH NP/Chitosan
10	oral	Humulin R-PLGA-PEG-	200	2194.26 ± 759.69	1.87 ± 0.65
		COOH NP-Lectin Conjugate

^aCalculated based on trapezoidal treatment of the serum Humulin R concentration (mU/L) vs time (hour) for both IV, IP and oral administration groups.
^bf_oral= (AUC_{0-20.5 h oral}/dose_oral)/(AUC_{0-7 h IV}/dose_IV). f_IP= (AUC_{0-7 h IP}/dose_IP)/(AUC_{0-7 h IV}/dose_IV). Mice were grouped to make average body weight to be 30 g in each group.

Humulin R Release Study
The release of Humulin from NPs and MPs was measured by incubating aliquots containing 2-5 mg of Humulin R-containing particles in 1.0 mL of 1×PBS at 37°. Measurements were conducted in triplicate. The supernatant was collected after centrifugation of the particle suspension at 14000 g for 5 min and analyzed with a BCA protein assay. The amount of Humulin R was calculated based on a standard curve generated with Humulin R stock solution.
Other embodiments of the invention will be apparent to those skilled in the art from a consideration of the specification or practice of the invention disclosed herein. It is intended that the specification and examples be considered as exemplary only, with the true scope and spirit of the invention being indicated by the following claims.

Claims

What is claimed is:

1. An oral composition comprising:

a plurality of nanoparticles having a diameter between about 10 nm and 1,000 nm,

the nanoparticles comprising

amphiphilic block co-polymer core encapsulating a therapeutic, diagnostic, or prophylactic agent, and

a mucoadhesive coating, the coating being present on the surface of the core and interacting with the amphiphilic block co-polymer through one or more of covalent interactions, affinity interactions, metal coordination, physical adsorption, host-guest interactions, and hydrogen bonding interactions, to form a coated nanoparticle; and

a pharmaceutically acceptable carrier.

2. The composition of claim 1, further comprising a targeting agent disposed under the mucoadhesive coating.

3. The composition of claim 2, further comprising an intermediate layer disposed between the targeting agent and the mucoadhesive coating.

4.-5. (canceled)

6. The composition of claim 3, wherein the intermediate layer comprises a biodegradable polymer.

7. The composition of claim 2, wherein the targeting agent is disposed at a surface portion of the polymer core.

8. The composition of claim 2, wherein the targeting agent is disposed at a surface portion of the polymer core and is also disposed at an interior portion of the polymer core.

9. The composition of claim 2, wherein the targeting agent is selected from the group consisting of nucleic acid aptamers, growth factors, hormones, cytokines, interleukins, antibodies, integrins, fibronectin receptors, p-glycoprotein receptors, and cell binding sequences.

10.-11. (canceled)

12. The composition of claim 1, wherein the mucoadhesive coating interacts with the core through hydrogen bonding interactions, covalent interactions, or a combination thereof.

13. The composition of claim 1, wherein the core comprises PEGylated poly(lactic acid).

14. The composition of claim 1, wherein the core comprises poly(alkylene glycol).

15. The composition of claim 14, wherein the poly(alkylene glycol) is selected from the group consisting of poly(ethylene glycol) and poly(propylene glycol).

16. The composition of claim 14, wherein the poly(alkylene glycol) has a molecular weight between about 100 and about 7000 Daltons.

17.-20. (canceled)

21. The composition of claim 1, wherein the amphiphilic block co-polymer comprises a biodegradable polymer is selected from poly(arylates), poly(anhydrides), poly(hydroxy acids), polyesters, poly(lactic acid), poly(glycolic acid), poly(ortho esters), polycarbonates, poly(propylene fumerates), poly(caprolactones), polyamides, polyphosphazenes, polyamino acids, polyethers, polyacetals, polylactides, polyhydroxyalkanoates, polyglycolides, polyketals, polyesteramides, poly(dioxanones), polyhydroxybutyrates, polyhydroxyvalyrates, polycarbonates, polyorthocarbonates, poly(vinyl pyrrolidone), biodegradable polycyanoacrylates, polyalkylene oxalates, polyalkylene succinates, poly(malic acid), poly(methyl vinyl ether), poly(ethylene imine), poly(acrylic acid), poly(maleic anhydride), biodegradable polyurethanes, polysaccharides, PEG-functionalized derivatives of the above, co-polymers of the above, adducts of the above, and mixtures of the above.

22. The composition of claim 1, wherein the amphiphilic block co-polymer comprises a non-biodegradable polymer is selected from the group consisting of polystyrene, polyesters, non-biodegradable polyurethanes, polyureas, poly(vinyl alcohol), polyamides, poly(tetrafluoroethylene), poly(ethylene vinyl acetate), polypropylene, polyacrylate, non-biodegradable polycyanoacrylates, polymethacrylate, poly(methyl methacrylate), polyethylene, polypyrrole, polyanilines, polythiophene, poly(ethylene oxide), PEG-functionalized derivatives of the above, co-polymers of the above, adducts of the above, and mixtures of the above.

23. The composition of claim 1, wherein the coating comprises poly(ethylene imine), lecithin, polycarboxylic acids, poly(acrylic acids), polysaccharides, polypeptides, co-polymers of the above, or any combination of the above.

24. The composition of claim 1, wherein the coating comprises chitosan, lectin, or both.

25. The composition of claim 1, wherein the coating is a block co-polymer having a mucoadhesive block and a block that is adapted to participate in an interaction selected from affinity interactions, metal coordination, physical adsorption, host-guest interactions, and hydrogen bonding interactions.

26. The composition of claim 1, wherein the therapeutic, diagnostic, or prophylactic agent is a biomolecule, bioactive agent, small molecule, drug, protein, vaccine, or polynucleotide.

27. (canceled)

28. The composition of claim 26, wherein the therapeutic agent is a protein.

29. The composition of claim 28, wherein the therapeutic agent is insulin.

30.-90. (canceled)

91. The composition of claim 14, wherein the poly(alkylene glycol) is terminated with a carboxyl group.

92. The composition of claim 23, wherein the polysaccharide comprises chitosan.

93. The composition of claim 23, wherein the polypeptide comprises a lectin.