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  Pharmaceutical Patents  

 

Title:  Controlled release nanoparticle having bound oligonucleotide for targeted delivery
United States Patent: 
7,727,969
Issued: 
June 1, 2010

Inventors:
 Farokhzad; Omid C. (Boston, MA), Jon; Sang Yong (Cambridge, MA), Langer; Robert S. (Newton, MA)
Assignee:
  Massachusetts Institute of Technology (Cambridge, MA)
Appl. No.:
 10/456,978
Filed:
 June 6, 2003


 

Executive MBA in Pharmaceutical Management, U. Colorado


Abstract

The present invention relates to a conjugate that includes a nucleic acid ligand bound to a controlled release polymer system, a pharmaceutical composition that contains the conjugate, and methods of treatment using the conjugate. The controlled release polymer system includes an agent such as a therapeutic, diagnostic, prognostic, or prophylactic agent. The nucleic acid ligand that is bound to the controlled release polymer system, binds selectively to a target, such as a cell surface antigen, and thereby delivers the controlled release polymer system to the target.

Description of the Invention

SUMMARY OF THE INVENTION

The present invention relates to a conjugate that includes a nucleic acid ligand bound to a controlled release polymer system, a pharmaceutical composition that contains the conjugate, and methods of treatment using the conjugate. The controlled release polymer system includes an agent such as a therapeutic, diagnostic, prognostic, or prophylactic agent. The nucleic acid ligand that is bound to the controlled release polymer system, binds selectively to a target, such as a cell surface antigen, and thereby delivers the controlled release polymer system to the target. The conjugate of the invention can be used to deliver an agent to a target cell by contacting the target cell with the conjugate.

Particular developmental stages of tissues are typically marked by specific cell surface antigens or express particular cell surface antigens significantly more frequently than tissues in another developmental stage. In one embodiment, the conjugate binds to a cell surface antigen specific to or more frequently expressed in a tissue in a specific developmental stage.

Particular disease states of a tissue, such as cancer, are typically marked by specific cell surface antigens or express a particular cell surface antigen more frequently than normal tissue. In one embodiment, the conjugate binds to a cell surface antigen specific to or more frequently expressed in a diseased state of a tissue.

In anther embodiment, a conjugate of the invention can be used to treat, diagnose or predict the outcome of a patient with cancer. The method involves administering to the patient a conjugate that comprises a nucleic acid ligand bound to a controlled release polymer system, wherein the nucleic acid ligand selectively binds to tumor cells.

The nucleic acid ligands are DNA or RNA oligonucleotides or modified DNA or RNA oligonucleotides that fold by intramolecular interactions into unique conformations. Like antibodies, nucleic acid ligands can be prepared that bind to target antigens with high specificity and affinity and thus can be used as targeting vehicles. However, the use of nucleic acid ligands as targeting vehicles has several advantages over antibodies. For example, nucleic acid ligands with a high affinity for a target can be prepared through a chemical process called Systemic Evolution of Ligands by Exponential Enrichment (SELEX), or a variation thereof. Nucleic acid ligands isolated using the SELEX process are relatively small as compared to antibodies, lack immunogenicity, and have been shown to have better tumor/plasma distribution, and are expected to have better tumor penetration potential. Moreover, since the SELEX process is a completely chemical process that does not involve animals, nucleic acid ligands can be prepared that bind to any target regardless of the toxicity or immunogenicity of the target. Furthermore, a nucleic acid ligand identified through the SELEX process can be synthesized using chemical oligonucleotide synthesis which has been shown to scale-up well so nucleic acid ligands can be prepared with little or no variation from batch to batch in binding affinity.

DETAILED DESCRIPTION OF THE INVENTION

A novel class of compounds known as nucleic acid ligands, for example aptamers, have recently been developed which rival antibodies in their potential for therapeutic and diagnostic use in many areas of medicine including oncology. Nucleic acid ligands are DNA or RNA oligonucleotides or modified DNA or RNA oligonucleotides that fold by intramolecular interactions into unique conformations capable of binding to target antigens with high specificity and affinity. Nucleic acid ligands for any antigen can be isolated by the Systemic Evolution of Ligands by Exponential Enrichment (SELEX), a technique first described by Tuerk and Gold in 1990 (Tuerk and Gold, Science (1990), 249:505-510, the entire teachings of which are incorporated herein by reference). Since their original description, nucleic acid ligands have been generated to a wide variety of targets, including intracellular proteins, transmembrane proteins, such as CD40 (U.S. Pat. No. 6,171,795, the entire teachings of which are incorporated herein by reference), soluble proteins, such as immunoglobulin E (U.S. Pat. Nos. 5,686,592 and 5,629,155, the entire teachings of both patents are incorporated herein by reference) and small molecule drugs, such as caffeine and theophylline (U.S. Pat. No. 5,580,737, the entire teachings of which are incorporated herein by reference). To date more than 100 nucleic acid ligands have been isolated, and one nucleic acid ligand against the Vascular Endothelial Growth Factor (VEGF)165 protein is currently in Phase III clinical trials for the treatment of neovascular macular degeneration (U.S. Pat. Nos. 6,426,335, 6,168,778, 6,051,698, 5,859,228, 5,849,479 and 5,811,533, all of which are incorporated herein by reference in their entirety). However, despite their similarity to antibodies in binding selectivity, nucleic acid ligands have not yet been fully exploited as delivery vehicles for therapeutic, diagnostic, prognostic and prophylactic agents. In particular, a conjugate of a nucleic acid ligand and a controlled release polymer system has not yet been demonstrated.

The present invention relates to a conjugate that has a nucleic acid ligand bound to a controlled release polymer system. A controlled release polymer system, as used herein, is a polymer combined with an active agent, such as a therapeutic agent, a diagnostic agent, a prognostic, or prophylactic agent, so that the active agent is released from the material in a predesigned manner. For example, the active agent may be released in a constant manner over a predetermined period of time, it may be released in a cyclic manner over a predetermined period of time, or an environmental condition or external event may trigger the release of the active agent. The controlled release polymer system may include a polymer that is biologically degradable, chemically degradable, or both biologically and chemically degradable.

Examples of suitable polymers for controlled release polymer systems include, but are not limited to, poly(lactic acid), derivatives of poly(lactic acid), PEGylated poly(lactic acid), poly(lactic-co-glycolic acid), derivatives of poly(lactic-co-glycolic acid), PEGylated poly(lactic-co-glycolic acid), polyanhydrides, poly(ortho esters) derivatives of poly(ortho esters), PEGylated poly(ortho esters), poly(caprolactones), derivatives of poly(caprolactone), PEGylated poly(caprolactone), polylysine, derivatives of polylysine, PEGylated polylysine, poly(ethylene imine), derivatives of poly(ethylene imine), PEGylated poly(ethylene imine), poly(acrylic acid), derivatives of poly(acrylic acid), poly(urethane), derivatives of poly(urethane), and combinations thereof. In a preferred embodiment, the controlled release polymer system is a microsphere or a nanosphere.

As used herein, "degradable" polymers are those that, when introduced into cells, are broken down by the cellular machinery (biologically degradable) and/or by a chemical process, such as hydrolysis, (chemically degradable) into components that the cells can either reuse or dispose of without significant toxic effect on the cells. In preferred embodiments, the degradable polymers and their degradation byproducts are biocompatible.

The term "biocompatible" polymer, as used herein is intended to describe polymers 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 if they do not induce significant inflammation or other such significant adverse effects in vivo.

Biocompatible polymers can be categorized as biodegradable and non-biodegradable. Biodegradable polymers degrade in vivo as a function of chemical composition, method of manufacture, and implant structure. Synthetic and natural polymers can be used although synthetic polymers are preferred due to more uniform and reproducible degradation and other physical properties. Examples of synthetic polymers include polyanhydrides, polyhydroxyacids such as polylactic acid, polyglycolic acids and copolymers thereof, polyesters, polyamides, polyorthoesters, and some polyphosphazenes. Examples of naturally occurring polymers include proteins and polysaccharides such as collagen, hyaluronic acid, albumin and gelatin. Drug can be encapsulated within, throughout, and/or on the surface of the implant. Drug is released by diffusion, degradation of the polymer, or a combination thereof. There are two general classes of biodegradable polymers: those degrading by bulk erosion and those degrading by surface erosion. 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.

Other polymers such as polylactic acid, polyglycolic acid, and copolymers thereof have been commercially available as suture materials for a number of years and can be readily formed into devices for drug delivery.

Non-biodegradable polymers remain intact in vivo for extended periods of time (e.g., at least about one or more years). Drug loaded into the non-biodegradable polymer matrix is released by diffusion through the polymer's micropore lattice in a sustained and predictable fashion, which can be tailored to provide a rapid or a slower release rate by altering the percent drug loading, porosity of the matrix, and implant structure. Ethylene-vinyl acetate copolymer (EVAc) is an example of a nonbiodegradable polymer that has been used as a local delivery system for proteins and other micromolecules, as reported by Langer, R., and J. Folkman, Nature (London) 263:797-799 (1976). Others include polyurethanes, polyacrylonitriles, and some polyphosphazenes.

Cationic polymers have been widely used as transfection vectors due to the facility with which they condense and protect negatively charged strands of DNA. Amine-containing polymers such as poly(lysine) (Zauner et al., Adv. Drug Del. Rev., 30:97-113, 1998; Kabanov et al., Bioconjugate Chem., 6:7-20, 1995, the entire teachings of each of the foregoing references are incorporated herein by reference), poly(ethylene imine) (PEI) (Boussif et al., Proc. Natl. Acad. Sci. USA, 92:7297-7301, 1995, the entire teachings of which are incorporated herein by reference), and poly(amidoamine) dendrimers (Kukowska-Latallo et al., Proc. Natl. Acad. Sci. USA, 93:4897-4902, 1996; Tang et al., Bioconjugate Chem. 7:703-714, 1996; Haensler et al., Bioconjugate Chem., 4:372-379, 1993; the entire teachings of each of the foregoing references are incorporated herein by reference) are positively-charged at physiological pH, form ion pairs with nucleic acids, and mediate transfection in a variety of cell lines.

Degradable polyesters bearing cationic side chains have also been developed (Putnam et al., Macromolecules, 32:3658-3662, 1999; Barrera et al., J. Am. Chem. Soc., 115:11010-11011, 1993; Kwon et al., Macromolecules, 22:3250-3255, 1989; Lim et al., J. Am. Chem. Soc., 121:5633-5639, 1999; Zhou et al., Macromolecules, 23:3399-3406, 1990, the entire teachings of each of the foregoing references are incorporated herein by reference). Examples of these polyesters include poly(L-lactide-co-L-lysine) (Barrera et al., J. Am. Chem. Soc., 115:11010-11011, 1993; the entire teachings of which are incorporated herein by reference), poly(serine ester) (Zhou et al., Macromolecules, 23:3399-3406, 1990, the entire teaching of each of the foregoing references are incorporated herein by reference), poly(4-hydroxy-L-proline ester) (Putnam et al., Macromolecules, 32:3658-3662, 1999; Lim et al., J. Am. Chem. Soc., 121:5633-5639, 1999, the entire teachings of each of the foregoing references are incorporated herein by reference). Poly(4-hydroxy-L-proline ester) was recently demonstrated to condense plasmid DNA through electrostatic interactions, and to mediate gene transfer (Putnam et al., Macromolecules, 32:3658-3662, 1999; Lim et al., J. Am. Chem. Soc., 121:5633-5639, 1999, the entire teachings of each of the foregoing references are incorporated herein by reference). Importantly, these new polymers are significantly less toxic than poly(lysine) and PEI, and they degrade into non-toxic metabolites.

The term "nucleic acids," or "oligonucleotides," as used herein, refers to a polymer of nucleotides. Typically, a nucleic acid comprises at least three nucleotides. The polymer may include natural nucleosides (i.e., adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine, deoxyguanosine, and deoxycytidine) or modified nucleosides. Examples of modified nucleotides include base modified nucleoside (e.g., aracytidine, inosine, isoguanosine, nebularine, pseudouridine, 2,6-diaminopurine, 2-aminopurine, 2-thiothymidine, 3-deaza-5-azacytidine, 2'-deoxyuridine, 3-nitorpyrrole, 4-methylindole, 4-thiouridine, 4-thiothymidine, 2-aminoadenosine, 2-thiothymidine, 2-thiouridine, 5-bromocytidine, 5-iodouridine, inosine, 6-azauridine, 6-chloropurine, 7-deazaadenosine, 7-deazaguanosine, 8-azaadenosine, 8-azidoadenosine, benzimidazole, M1-methyladenosine, pyrrolo-pyrimidine, 2-amino-6-chloropurine, 3-methyl adenosine, 5-propynylcytidine, 5-propynyluridine, 5-bromouridine, 5-fluorouridine, 5-methylcytidine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, O(6)-methylguanine, and 2-thiocytidine), chemically or biologically modified bases (e.g., methylated bases), modified sugars (e.g., 2'-fluororibose, 2'-aminoribose, 2'-azidoribose, 2'-O-methylribose, L-enantiomeric nucleosides arabinose, and hexose), modified phosphate groups (e.g., phosphorothioates and 5'-N-phosphoramidite linkages), and combinations thereof. Natural and modified nucleotide monomers for the chemical synthesis of nucleic acids are readily available (e.g. see, www.trilinkbiotech.com, www.appliedbiosystems.com, www.biogenex.com or www.syngendna.com).

As used herein, "nucleic acid ligand" is a non-naturally occurring nucleic acid that binds selectively to a target. The nucleic acid that forms the nucleic acid ligand may be composed of naturally occurring nucleosides, modified nucleosides, naturally occurring nucleosides with hydrocarbon linkers (e.g., an alkylene) or a polyether linker (e.g., a PEG linker) inserted between one or more nucleosides, modified nucleosides with hydrocarbon or PEG linkers inserted between one or more nucleosides, or a combination of thereof. In one embodiment, nucleotides or modified nucleotides of the nucleic acid ligand can be replaced with a hydrocarbon linker or a polyether linker provided that the binding affinity and selectivity of the nucleic acid ligand is not substantially reduced by the substitution (e.g., the dissociation constant of the nucleic acid ligand for the target should not be greater than about 1.times.10.sup.-6 M). The target molecule of a nucleic acid ligand is a three dimensional chemical structure that binds to the nucleic acid ligand. However, the nucleic acid ligand is not simply a linear complementary sequence of a nucleic acid target but may include regions that bind via complementary Watson-Crick base pairing interrupted by other structures such as hairpin loops). In the present invention, the targets include peptide, polypeptide, carbohydrate and nucleic acid molecules. In one embodiment, the nucleic acid ligand binds to a cell or tissue in a specific developmental stage or a specific disease state. A preferred target is an antigen on the surface of a cell, such as a cell surface receptor, an integrin, a transmembrane protein, and ion channel or a membrane transport protein. More preferably, the target is a tumor-marker. A tumor-marker is an antigen that is present in a tumor that is not present in normal tissue or an antigen that is more prevalent in a tumor than in normal tissue.

Nucleic acid ligands may be prepared by any method. However, a preferred method of preparing nucleic acid ligands is to identify nucleic acid ligands from a candidate mixture of nucleic acids. Systemic Evolution of Ligands by Exponential Enrichment (SELEX), or a variation thereof, is a commonly used method of identifying nucleic acid ligands that bind to a target from a candidate mixture of nucleic acids.

The SELEX process for obtaining nucleic acid ligands is described in U.S. Pat. No. 5,567,588, the entire teachings of which are incorporated herein by reference. Briefly, the basic SELEX process may be defined by the following series of steps:

1) A candidate mixture of nucleic acids of differing sequence is prepared. The candidate mixture generally includes regions of fixed sequences (i.e., each of the members of the candidate mixture contains the same sequences in the same location) and regions of randomized sequences. The fixed sequence regions are selected either: a) to assist in the amplification steps described below; b) to mimic a sequence known to bind to the target; or c) to enhance the potential of a given structural arrangement of the nucleic acids in the candidate mixture. The randomized sequences can be totally randomized (i.e., the probability of finding a base at any position being one in four) or only partially randomized (e.g., the probability of finding a base at any location can be selected at any level between 0 and 100 percent).

2) The candidate mixture is contacted with a selected target under conditions favorable for binding between the target and members of the candidate mixture. Under these circumstances, the interaction between the target and the nucleic acids of the candidate mixture can be considered as forming nucleic acid-target pairs between the target and the nucleic acids having the strongest affinity for the target.

3) The nucleic acids with the highest affinity for the target are partitioned from those nucleic acids with lesser affinity to the target. Because only an extremely small number of sequences (and possibly only one molecule of nucleic acid) corresponding to the highest affinity nucleic acids exist in the candidate mixture, it is generally desirable to set the partitioning criteria so that a significant amount of the nucleic acids in the candidate mixture (approximately 0.1%-10%) is retained during partitioning.

4) Those nucleic acids selected during partitioning as having the relatively higher affinity to the target are then amplified to create a new candidate mixture that is enriched in nucleic acids having a relatively higher affinity for the target.

5) By repeating the partitioning and amplifying steps above, the newly formed candidate mixture contains fewer and fewer unique sequences, and the average degree of affinity of the nucleic acid mixture to the target will generally increase. Taken to its extreme, the SELEX process will yield a candidate mixture containing one or a small number of unique nucleic acids representing those nucleic acids from the original candidate mixture having the highest affinity to the target molecule. Preferably, the nucleic acid ligands identified will have a dissociation constant with the target of about 1.times.10.sup.-6 M or less. Typically, the dissociation constant of the nucleic acid ligand and the target will be in the range of between about 1.times.10.sup.-8 M and about 1.times.10.sup.-12 M.

Nucleic acid ligands that bind selectively to any antigen can be isolated by the SELEX process, or a variation thereof, provided that the antigen can be used as a target in the SELEX process. For example, nucleic acid ligands that bind to a protein, a carbohydrate or a nucleic acid can be prepared. In one embodiment, nucleic acid ligands can be prepared and used in the conjugates of the invention that bind to proteins, such as tumor-markers, integrins, cell surface receptors, transmembrane proteins, intercellular proteins, ion channels, membrane transporter protein, enzymes, antibodies, and chimeric proteins. In another embodiment, nucleic acid ligands can be prepared and used in the conjugates of the invention that bind to carbohydrate, such as glycoproteins, sugar residues (e.g., monosaccharides, disaccharides and polysaccharides) and glycocalyx (i.e., the carbohydrate-rich peripheral zone on the outside surface of most eucaryotic cells). In another embodiment, nucleic acid ligands can be prepared and used in the conjugates of the invention that bind to a nucleic acid, such as DNA nucleic acids, RNA nucleic acids, modified DNA nucleic acids, modified RNA nucleic acids or nucleic acids that include any combination of DNA, RNA, modified DNA, and modified RNA. In a preferred embodiment, the nucleic acid ligand used in the conjugates of the invention binds to a cell surface antigen.

Preferably, the nucleic acid ligand is resistant to endonuclease and exonuclease degradation. Typically, nucleic acid ligands that includes one or more modified nucleotide exhibit improved resistant to endo- and exonuclease degradation. A particularly preferred nucleic acid ligand is a spiegelmer which is composed of L-enantiomeric nucleotides and is resistant to endo- and exonucleases. The nucleic acid ligands used in the conjugates of the invention can also include one or more internal nucleotides at the 3'-end, 5'-end, or at both the 3'- and 5'-end that is inverted to yield a 3'-3' or a 5'-5' nucleotide linkage.

Nucleic acid ligands can increase the specificity of the controlled release polymer system that contains a therapeutic, diagnostic and prognostic agents, thus increasing their efficacy while decreasing their toxicity. For example, in a conjugate designed for a therapeutic application, a nucleic acid ligand can be conjugated with a controlled release polymer system that includes a toxin, radionuclide or chemotherapeutic drug; this conjugated nucleic acid ligand may be simplistically viewed as a guided missile with the nucleic acid ligand as the guidance system and the drug as the warhead. In addition, nucleic acid ligand conjugated with radionuclides or metallic tracers can be used for proton emission (PET) and nuclear magnetic resonance (NMR) imaging for in vivo diagnosis and determination of the location of, for example, metastases.

The conjugate of the invention can also include an agent for localization that aids in positioning the conjugate in the vicinity of the target cell or tissue.

For example, a magnetic particle may be attached to the conjugate as a localizing agent. The conjugate can then be administered to the patient intravenous, for example, and external magnets can be positioned so that a magnetic field is created within the body at the site of the target tissue. The magnetic particle that is attached to the conjugate is then drawn to the magnetic field and retained there until the magnet is removed. In one embodiment, the magnetic particle includes elemental iron.

The agents to be incorporated in the controlled release polymer system and delivered to a target cell or tissue by a conjugate 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 agent may be a small molecule, organometallic compound, nucleic acid, protein, peptide, polynucleotide, metal, an isotopically labeled chemical compound, drug, vaccine, immunological agent, etc.

In a preferred 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 a particularly preferred embodiment, 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.

"Small molecule": As used herein, the term "small molecule" refers to organic compounds, whether naturally-occurring or artificially created (e.g., via chemical synthesis) that have relatively low molecular weight and that are not proteins, polypeptides, or nucleic acids. Typically, small molecules have a molecular weight of less than about 2500 g/mol. Also, small molecules typically have multiple carbon-carbon bonds. Small molecules include, but are not limited to, an antibiotic, an antiviral, an antiparasitic agent, an anti-cancer agent, a radionuclide, an anticoagulant, an analgesic agent, an anesthetic agent, an ion channel potentiator, an ion channel inhibitor, an anti-inflammatory and combinations thereof. 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.

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. According to the present invention, a "peptide" or "protein" comprises a string of at least three amino acids linked together by peptide bonds. The terms "protein" and "peptide" may be used interchangeably. 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.

In another embodiment, the agent 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 promotor complementary region, a repressor complementary region, an enhancer complementary region, and combinations thereof. A promotor complementary region, a repressor complementary region, an enhancer complementary region can be fully complementary or partially complementary to the DNA promotor region, repressor region, an enhancer region of a gene for which it is desirable to modulate expression.

In a preferred embodiment of the present invention, the agent to be delivered may be a mixture of agents. For example, a local anesthetic may be delivered in combination with an anti-inflammatory agent such as a steroid. Local anesthetics may also be administered with vasoactive agents such as epinephrine. To give another 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, the controlled release polymer systems of the conjugates of the invention include a fluorescent molecule as a diagnostic agent.

Other diagnostic agents include gases; metals; commercially available imaging agents used in positron emissions tomography (PET), computer assisted tomography (CAT), single photon emission computerized tomography, x-ray, fluoroscopy, and magnetic resonance imaging (MRI); and contrast agents. Examples of suitable materials for use as contrast agents in MRI include gadolinium chelates, as well as iron, magnesium, manganese, copper, and chromium. Examples of materials useful for CAT and x-ray imaging include iodine-based materials.

The controlled release polymer systems used in the conjugates of the invention may include radionuclides as either therapeutic, diagnostic, or prognostic agents. Among the radionuclides used, gamma-emitters, positron-emitters, and X-ray emitters are suitable for diagnostic and/or therapy, while beta emitters and alpha-emitters may also be used for therapy. Suitable radionuclides for forming the conjugate of the invention include, but are not limited to, .sup.123I, .sup.125I, .sup.130I, .sup.131I, .sup.133I, .sup.135I, .sup.47Sc, .sup.72As, .sup.72Se, .sup.90Y, .sup.88Y, .sup.97Ru, .sup.100Pd, .sup.101mRh, .sup.119Sb, .sup.128Ba, .sup.197Hg, .sup.211At, .sup.212Bi, .sup.212Pb, .sup.109Pd, .sup.111In, .sup.67Ga, .sup.68Ga, .sup.67Cu, .sup.75Br, .sup.77Br, .sup.99mTc, .sup.14C, .sup.13N, .sup.15O, .sup.32P, .sup.33P, and .sup.18F.

In another embodiment, the controlled release polymer systems used in the conjugates of the invention may include a diagnostic or prognostic agent used in magnetic resonance imaging (MRI), such as iron oxide particles or gadolinium complexes. Gadolinium complexes that have been approved for clinical use include gadolinium chelates with DTPA, DTPA-BMA, DOTA and HP-DO3A which are reviewed in Aime, et al., Chemical Society Reviews (1998), 27:19-29, the entire teachings of which are incorporated herein by reference.

Prophylactic agents that can be included in the conjugates of the invention include, but are not limited to, antibiotics, nutritional supplements, and vaccines. 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 anthracis, 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.

When conjugating nucleic acid ligands to controlled release polymer systems, it is desirable to have a polymer system which can be efficiently linked to the negatively charged nucleic acid ligands using simple chemistry without adversely affecting the 3-dimensional characteristic and conformation of the nucleic acid ligands, which is important for its target recognition. It is also desirable that a delivery vehicle should be able to avoid uptake by the mononuclear phagocytic system after systemic administration so that it is able to reach specific tissues and cells in the body. Polymers that are suitable for the controlled release polymer system include, but are not limited to, poly(lactic acid), poly(lactic-co-glycolic acid), polyanhydrides, poly(ortho esters)poly(caprolactone), polylysine, poly(ethylene imine), poly(acrylic acid), poly(urethane), and derivatives thereof. Preferred derivatives are polymers that have been PEGylated (i.e., covalently linked to polyethylene glycol), such as PEGylated poly(lactic acid), PEGylated poly(lactic-co-glycolic acid), PEGylated poly(caprolactone), PEGylated poly(ortho esters), PEGylated polylysine, and PEGylated poly(ethylene imine). In some instances, polymers that have a positive surface charge may result in a non-specific nucleic acid ligand-polymer interaction. This may result in decrease specific binding properties of some of the nucleic acid ligands bound to the controlled release polymer system. Therefore, in a preferred embodiment, the polymer carries a neutral to negative charge on its surface. Examples of polymer particles that carry a neutral to negative surface charge include, but are not limited to, polysebacic anhydride (PSA) and poly(lactic acid).

Positively charged polymers such as poly(ethylene imine) or polylysine are typically used for nucleic acid encapsulation and gene delivery. However, as discussed above, positively charged polymers may interact with the negatively charged nucleic acid ligands and adversely affect their 3-dimensional characteristics and conformation and thereby disrupt target recognition of some of the nucleic acid ligands bound to the controlled release polymer system. One method of decreasing the charge interaction between the controlled release polymer system and nucleic acid ligand is to create a hydrophilic layer on the surface of the polymer particles by attaching polyethyleneglycol (PEG) to the surface of the polymer to shield the particles from interacting with nucleic acid ligands. In addition, many investigators have reported that the hydrophilic PEG group will increase plasma half-life by decreasing the uptake of the polymer by the phagocytic system while decreasing transfection/uptake efficiency by cells. Interestingly, lower transfection/uptake efficiency as a result of PEG placement on the surface of nanoparticles is a favorable factor because it will potentially decrease non-specific transfection/uptake, while the presence of conjugated nucleic acid ligands on the polymer surface will increase transfection/uptake efficiency in targeted cells. Therefore, a dramatic decrease in transfection/uptake efficiency on cells which do not express the target of the nucleic acid ligand and a relatively less severe reduction in transfection/uptake efficiency in cells which do express these targets is expected.

The controlled release polymer system can be attached to the nucleic acid ligand directly or via a linker. When the nucleic acid ligand is attached directly to the controlled release polymer system, it is preferably attached by forming a covalent bond between a functional group on the nucleic acid ligand, such as an amine, a thiol or a hydroxy group, and a functional group on the controlled release polymer system, such as a carboxylic acid, an anhydride, a maleimide, an aldehyde group, etc. In a preferred embodiment, a linker is used to attach the controlled release polymer system to the nucleic acid ligand. A linker typically has a functional group that can form a bond with the nucleic acid ligand and a functional group that can form a bond with the controlled release polymer system. The functional groups of the linker can be separated by one or more atoms that are relatively non-reactive, such as an alkylene or a polyether (e.g., --{O(CH.sub.2).sub.m}.sub.n--). In one embodiment, either the bond between the linker and the nucleic acid ligand, or the bond between the linker and the controlled release polymer system, or both bonds are covalent. In addition, monomers are available for chemical synthesis of nucleic acids that have a linker attached to the nucleotide base or sugar (e.g., see www.trilinkbiotech.com). These monomers can be incorporated into the nucleic acid ligand during chemical synthesis.

Alternatively, the controlled release polymer system can be attached to the nucleic acid ligand directly or indirectly via a non-covalent interactions. Non-covalent interactions include but are not limited to the following:

1) Charge Interactions: For example, the controlled release polymer system may have a cationic surface or may be reacted with a cationic polymer, such as poly(lysine) or poly(ethylene imine), to provide a cationic surface. The polymer surface can then bind via charge interactions with a negatively charged nucleic acid ligand. One end of the nucleic acid ligand is, typically, attached to a negatively charged polymer (e.g., a poly(carboxylic acid)) or an additional oligonucleotide sequence that can interact with the cationic polymer surface without disrupting the binding affinity of the nucleic acid ligand for its target.

2) Affinity Interactions: For example, biotin may be attached to the surface of the controlled release polymer system and streptavidin may be attached to the nucleic acid ligand; or conversely, biotin may be attached to the nucleic acid ligand and the streptavidin may be attached to the surface of the controlled release polymer system. The biotin group and streptavidin are typically attached to the controlled release polymer system or to the nucleic acid ligand via a linker, such as an alkylene linker or a polyether linker. Biotin and streptavidin bind via affinity interactions, thereby binding the controlled release polymer system to the nucleic acid ligand.

3) Metal Coordination: For example, a polyhistidine may be attached to one end of the nucleic acid ligand, and a nitrilotriacetic acid can be attached to the surface of the controlled release polymer system. A metal, such as Ni.sup.+2, will chelate the polyhistidine and the nitrilotriacetic acid, thereby binding the nucleic acid ligand to the controlled release polymer system.

4) Physical Adsorption: For example, a hydrophobic tail, such as polymethacrylate or an alkyl group having at least about 10 carbons, may be attached to one end of the nucleic acid ligand. The hydrophobic tail will adsorb onto the surface of a hydrophobic controlled release polymer system, such as a controlled release polymer system made of or coated with a polyorthoester, polysebacic anhydride, or polycaprolactone, thereby binding the nucleic acid ligand to the controlled release polymer system.

5) Host-Guest Interactions: For example, a macrocyclic host, such as cucurbituril or cyclodextrin, may be attached to the surface of the controlled release polymer system and a guest group, such as an alkyl group, a polyethylene glycol, or a diaminoalkyl group, may be attached to the nucleic acid ligand; or conversely, the host group may be attached to the nucleic acid ligand and the guest group may be attached to the surface of the controlled release polymer system. In one embodiment, the host and/or the guest molecule may be attached to the nucleic acid ligand or the controlled release polymer system via a linker, such as an alkylene linker or a polyether linker.

6) Hydrogen Bonding Interactions: For example, an oligonucleotide having a particular sequence may be attached to the surface of the controlled release polymer system, and an essentially complementary sequence may be attached to one or both ends of the nucleic acid ligand such that it does not disrupt the binding affinity of the nucleic acid ligand for its target. The nucleic acid ligand will then bind to the controlled release polymer system 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.

Once the inventive conjugates have been prepared, they may be combined with pharmaceutical acceptable carriers to form a pharmaceutical composition. As would be appreciated by one of skill in this art, the carriers may be chosen based on the route of administration as described below, the location of the target issue, the drug being delivered, the time course of delivery of the drug, etc.

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.TM. 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, as well as coloring agents, releasing agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the composition, according to the judgment of the formulator.

The pharmaceutical compositions of this 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). In certain embodiments parenteral routes are preferred since they avoid contact with the digestive enzymes that are found in the alimentary canal. According to such embodiments, inventive 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).

Injectable preparations, for example, sterile injectable aqueous or oleaginous suspensions may be formulated according to the known art using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation may also be a sterile injectable solution, suspension, or emulsion in a nontoxic parenterally acceptable diluent or solvent, for example, as a solution in 1,3-butanediol. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution, U.S.P., and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose any bland fixed oil can be employed including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid are used in the preparation of injectables. In a particularly preferred embodiment, the inventive conjugate is suspended in a carrier fluid comprising 1% (w/v) sodium carboxymethyl cellulose and 0.1% (v/v) TWEEN.TM. 80. The injectable formulations can be sterilized, for example, by filtration through a bacteria-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable medium prior to use.

Compositions for rectal or vaginal administration are preferably suppositories which can be prepared by mixing the inventive conjugate with suitable non-irritating excipients or carriers such as cocoa butter, polyethylene glycol, or a suppository wax which are solid at ambient temperature but liquid at body temperature and therefore melt in the rectum or vaginal cavity and release the inventive conjugate.

Dosage forms for topical or transdermal administration of an inventive pharmaceutical composition include ointments, pastes, creams, lotions, gels, powders, solutions, sprays, inhalants, or patches. The inventive conjugate is admixed under sterile conditions with a pharmaceutically acceptable carrier and any needed preservatives or buffers as may be required. Ophthalmic formulations, ear drops, and eye drops are also contemplated as being within the scope of this invention. The ointments, pastes, creams, and gels may contain, in addition to the inventive conjugates of this invention, excipients such as animal and vegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicic acid, talc, and zinc oxide, or mixtures thereof. Transdermal patches have the added advantage of providing controlled delivery of a compound to the body. Such dosage forms can be made by dissolving or dispensing the inventive conjugates in a proper medium. Absorption enhancers can also be used to increase the flux of the compound across the skin. The rate can be controlled by either providing a rate controlling membrane or by dispersing the inventive conjugates in a polymer matrix or gel.

Powders and sprays can contain, in addition to the inventive conjugates 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.

When administered orally, the inventive conjugates are preferably, but not necessarily, encapsulated. A variety of suitable encapsulation systems are known in the art ("Microcapsules and Nanoparticles in Medicine and Pharmacy," Edited by Doubrow, M., CRC Press, Boca Raton, 1992; Mathiowitz and Langer J. Control. Release 5:13, 1987; Mathiowitz et al. Reactive Polymers 6:275, 1987; Mathiowitz et al. J. Appl. Polymer Sci. 35:755, 1988; Langer Acc. Chem. Res. 33:94, 2000; Langer J. Control. Release 62:7, 1999; Uhrich et al. Chem. Rev. 99:3181, 1999; Zhou et al. J. Control. Release 75:27, 2001; and Hanes et al. Pharm. Biotechnol. 6:389, 1995). Preferably the inventive conjugates are encapsulated within biodegradable polymeric microspheres or liposomes. Examples of natural and synthetic polymers useful in the preparation of biodegradable microspheres include carbohydrates such as alginate, cellulose, polyhydroxyalkanoates, polyamides, polyphosphazenes, polypropylfumarates, polyethers, polyacetals, polycyanoacrylates, biodegradable polyurethanes, polycarbonates, polyanhydrides, polyhydroxyacids, poly(ortho esters), and other biodegradable polyesters. Examples of lipids useful in liposome production include phosphatidyl compounds, such as phosphatidylglycerol, phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine, sphingolipids, cerebrosides, and gangliosides.

Pharmaceutical compositions for oral administration can be liquid or solid. Liquid dosage forms suitable for oral administration of inventive compositions include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups, and elixirs. In addition to an encapsulated or unencapsulated conjugate, 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 conjugate 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 conjugate 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 inventive conjugate to the patient being treated. As used herein, the "effective amount" of an inventive conjugate refers to the amount necessary to elicit the desired biological response. As will be appreciated by those of ordinary skill in this art, the effective amount of inventive conjugate may vary depending on such factors as the desired biological endpoint, the drug to be delivered, the target tissue, the route of administration, etc. For example, the effective amount of inventive conjugate 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 conjugates of the invention are preferably formulated 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 conjugate, 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 conjugates can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., ED.sub.50 (the dose is therapeutically effective in 50% of the population) and LD.sub.50 (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, LD.sub.50/ED.sub.50. 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.
 

Claim 1 of 23 Claims

1. A controlled release polymer nanoparticle, comprising: a therapeutic or diagnostic agent incorporated in the controlled release polymer nanoparticle; a nucleic acid ligand comprising an oligonucleotide bound to the surface of the controlled release polymer nanoparticle that selectively binds to a target cell or tissue, and thereby delivers the controlled release polymer nanoparticle to the target cell or tissue; wherein the controlled release polymer nanoparticle carries a neutral to negative charge on its surface, wherein the bound oligonucleotide has a folded three-dimensional conformation resulting from intramolecular interactions; and wherein the nucleic acid ligand is an aptamer.

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