This application claims benefit of U.S. application No. 61/668,446 filed on 7/5/2012 and U.S. application No. 61/791,650 filed on 3/15/2013, the entire disclosures of which are incorporated herein by reference.
Drawings
Figure 1 is a line graph showing the change in body weight of 4 groups of rats after each group had been subjected to a single IA injection of saline, free Dexamethasone Sodium Phosphate (DSP) or a sustained release composition.
Figure 2 is a line graph showing the change in clinical visual arthritis score for 4 groups of rats after each group had been subjected to a single IA injection of saline, free DSP or sustained release composition.
Figure 3 is a line graph showing the change in right ankle volume of 4 groups of rats after each group had been subjected to a single IA injection of saline, free DSP or a sustained release composition.
Fig. 4 is a line graph showing the change in left ankle volume of 4 groups of rats after each group had been subjected to a single IA injection of saline, free DSP or a sustained release composition.
Fig. 5 is a line graph showing the change in body weight of 3 groups of rats following four once daily (daily) IA injections of free DSP or sustained release composition.
Figure 6 is a line graph showing the change in clinical visual arthritis score for 3 groups of rats after each group had been subjected to four once daily IA injections of either free DSP or sustained release composition.
Figure 7 is a line graph showing the change in right ankle volume of 3 groups of rats after each group had been subjected to four once daily IA injections of either free DSP or a sustained release composition.
Fig. 8 is a line graph showing the change in left ankle volume of 3 groups of rats after each group had been subjected to four once daily IA injections of either free DSP or a sustained release composition.
Fig. 9 is a line graph showing the change in body weight (panel a) and the change in clinical arthritis score (panel b) of 3 groups of rats after five once daily IA injections of either free indomethacin or an indomethacin sustained release composition. The first arrow (day 19) indicates the first administration of indomethacin once daily for IA, and the second arrow (day 23) indicates the last administration of indomethacin once daily for IA.
Fig. 10 is a line graph showing the change in body weight (panel a) and the change in clinical arthritis score (panel b) of 3 groups of rats after two IA injections of free etanercept or a sustained release composition of etanercept. The first arrow (day 23) indicates a first administration of etanercept IA and the second arrow (day 26) indicates a second administration of etanercept IA.
Figure 11 is a line graph showing the change in body weight (panel a) and the change in clinical arthritis score (panel b) in 3 groups of rats after two IA injections of either free methotrexate or a methotrexate sustained release composition. The first arrow (day 23) indicates the first methotrexate IA administration and the second arrow (day 26) indicates the second methotrexate IA administration.
Detailed description of the invention
As disclosed herein, it has been found that administration of an effective amount of a sustained release composition described herein to a subject IA can advantageously reduce the signs and/or symptoms of arthritis in said subject. It has also been found that the arthritis treatment disclosed herein may require less frequent IA injections (as compared to previously known treatments). The arthritis treatment disclosed herein was also found to provide longer term relief from pain than previously known treatments. These findings are embodied in the methods, compositions and medicaments for treating arthritis described herein and in the use of the compositions for treating arthritis.
Definition of
As used above and throughout the disclosure, the following terms, unless otherwise indicated, shall be understood to have the following meanings.
As used herein, the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise.
As used herein, the term "liposome/liposomes" and related terms include multivesicular liposomes (MVLs), multilamellar vesicles (MLVs), or small or large unilamellar vesicles (ULVs). Liposomes are nano-sized and comprise a particle-forming component and a drug-carrying component. The particle-forming components form a closed lipid barrier that is substantially free of neutral lipids such as triglycerides. In certain embodiments, less than about 0.1% neutral lipids are present in the particle-forming component. In other embodiments, the particle-forming component is free of neutral lipids. The components of the carrier agent comprise a substantial amount of an aqueous medium that is substantially free of neutral lipids such as triglycerides, a non-aqueous phase (oil phase), a water-oil emulsion, or other mixtures containing a non-aqueous phase.
As used herein, the term "effective amount" refers to a dosage of a sustained release composition sufficient to alleviate symptoms and/or signs of arthritis, such as pain and ankylosis.
As used herein, the terms "treating" or "treated" include prophylactic (e.g., prophylactic), palliative, and curative methods, uses, or results. The term "treatment" may also refer to compositions, such as pharmaceutical compositions, or drugs.
In this application, treatment means a method of alleviating or delaying one or more effects or symptoms of arthritis. Treatment may also refer to methods of alleviating the underlying cause rather than just the symptoms. The treatment may be any alleviation and may be, but is not limited to: arthritis, complete elimination of signs or symptoms of arthritis. Treatment may include complete amelioration of arthritis as detected by known techniques. There are art-recognized methods for detecting arthritis and its symptoms. They include, but are not limited to: such as radiology, joint aspiration, blood tests (e.g., to detect rheumatoid factor or anti-CCP tests), or MRI. For example, a disclosed method is considered a treatment if one or more symptoms of arthritis in a subject are reduced by about 10% as compared to the subject or a control subject prior to treatment. Thus, the mitigation may be about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or any mitigation amount therebetween.
As used herein, preventing (preceding, preceding or preceding) means a method of arresting, delaying, preventing, avoiding, pre-arresting, terminating or hindering the onset, occurrence, severity or recurrence of arthritis. For example, a disclosed method is considered a prophylaxis if an individual susceptible to arthritis has reduced or delayed onset, occurrence, severity, or recurrence of arthritis or one or more symptoms of arthritis (e.g., pain, stiffness, fever, joint inflammation, or joint tenderness) as compared to a control individual susceptible to arthritis and not receiving treatment as disclosed herein. A disclosed method is also considered a prophylaxis if an individual susceptible to arthritis has a reduced or delayed onset, occurrence, severity, or recurrence of arthritis or one or more symptoms of arthritis as compared to the individual's progression prior to receiving treatment after receiving treatment disclosed herein. Thus, the reduction or delay in the onset, incidence, severity, or recurrence of arthritis may be about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or any reduction therebetween.
The term "individual" may refer to a vertebrate suffering from arthritis or a vertebrate believed to be in need of arthritis treatment. Individuals include warm-blooded animals, such as mammals, such as primates, and more preferably humans. A non-human primate is also a subject. The term subject includes domestic animals (such as cats, dogs, etc.), livestock (e.g., cows, horses, pigs, sheep, goats, etc.), and laboratory animals (e.g., mice, rabbits, rats, gerbils, guinea pigs, etc.). Thus, veterinary uses and pharmaceutical formulations are contemplated herein. The term "arthritis" refers to a joint disorder or condition involving inflammation of one or more joints. As used herein, the term "arthritis" encompasses various types and subtypes of arthritis of known or unknown origin and etiology, including, but not limited to: rheumatoid arthritis, osteoarthritis, infectious arthritis, psoriatic arthritis, gouty arthritis and lupus-associated arthritis.
Lipid mixture
The lipid mixture for the arthritis treatment described herein contains a solid lipid mixture (lipid mixture) in the form of a cake, film or powder.
In certain embodiments, the phospholipid and cholesterol, or mixture of phospholipids and cholesterol, are preformed into liposomes prior to further processing into a lipid mixture.
In certain embodiments, the phospholipid and cholesterol, or mixture of phospholipids and cholesterol, are not preformed into liposomes prior to further processing into a lipid mixture.
Lipid mixtures can be prepared from a variety of lipids capable of forming or incorporating monolayer or bilayer structures. The lipid mixtures provided herein comprise one or more phospholipids and cholesterol, which are substantially free of neutral lipids such as triglycerides. Examples of phospholipids include, but are not limited to: phosphatidyl Choline (PC), Phosphatidyl Glycerol (PG), Phosphatidyl Ethanolamine (PE), Phosphatidyl Serine (PS), Phosphatidic Acid (PA), Phosphatidyl Inositol (PI), Egg Phosphatidyl Choline (EPC), Egg Phosphatidyl Glycerol (EPG), Egg Phosphatidyl Ethanolamine (EPE), Egg Phosphatidyl Serine (EPS), lecithin acid (EPA), Egg Phosphatidyl Inositol (EPI), Soybean Phosphatidyl Choline (SPC), Soybean Phosphatidyl Glycerol (SPG), Soybean Phosphatidyl Ethanolamine (SPE), Soybean Phosphatidyl Serine (SPS), Soybean Phosphatidic Acid (SPA), Soybean Phosphatidyl Inositol (SPI), Dipalmitoylphosphatidylcholine (DPPC), 1, 2-dioleoyl-sn-glycerol-3-phosphatidyl choline (DOPC), dimyristoyl choline (DMPC), Dipalmitoylphosphatidylglycerol (DPPG), Dioleoylphosphatidylglycerol (DOPG), Dimyristoylphosphatidylglycerol (DMPG), cetylphosphorylcholine (HEPC), Hydrogenated Soy Phosphatidylcholine (HSPC), Distearoylphosphatidylcholine (DSPC), Distearoylphosphatidylglycerol (DSPG), Dioleoylphosphatidylethanolamine (DOPE), palmitoylstearoylphosphatidylcholine (PSPC), palmitoylstearoylphosphatidylglycerol (PSPG), Monooleoylphosphatidylethanolamine (MOPE), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (POPC), polyethylene glycol distearoylphosphatidylethanolamine (PEG-DSPE), Dipalmitoylphosphatidylserine (DPPS), 1, 2-dioleoyl-sn-glycero-3-phosphatidylserine (DOPS), Dimyristoylphosphatidylserine (DMPS), Distearoylphosphatidylserine (DSPS), dipalmitoylphosphatidic acid (DPPA), 1, 2-dioleoyl-sn-glycero-3-phosphatidic acid (DOPA), dimyristoylphosphatidic acid (DMPA), distearoylphosphatidic acid (DSPA), Dipalmitoylphosphatidylglycerol (DPPI), 1, 2-dioleoyl-sn-glycero-3-phosphatidylinositol (DOPI), Dimyristoylphosphatidylglycerol (DMPI), Distearoylphosphatidylinositol (DSPI). The lipid may be a mixture of one or more of the above lipids, or a mixture of one or more of the above lipids with one or more other lipids not listed above.
In an exemplary embodiment, the lipid mixture comprises a mixture of two phospholipids, such as DOPC or DOPG. In another embodiment, the lipid mixture comprises a mixture of phospholipids and cholesterol selected from the group consisting of DOPC, POPC, SPC, EPC, PEG-DSPE and DOPG. In another embodiment, the lipid mixture comprises a mixture of a first phospholipid which is DOPC, POPC, SPC or EPC and a second phospholipid which is PEG-DSPE or DOPG. Various exemplary compositions of lipid mixtures are disclosed in U.S. application Ser. No. 12/538,435, the teachings of which are incorporated herein by reference in their entirety.
In an exemplary embodiment, the lipid blend comprises DOPC, DOPG and cholesterol in a molar ratio of about (29.5% to 90%) (3% to 37.5%): (10% to 33%). In another embodiment, the ratio of DOPC to DOPG to cholesterol is about (56.25-72.5): (7.5-18.75): (20-25) in mole percent. For example, but not by way of limitation, the ratio of DOPC to DOPG to cholesterol may be about 67.5:7.5: 25. In another embodiment, the lipid blend (lipid cake mix) comprises from about 12 mole% to less than about 30 mole% cholesterol relative to the lipid blend. In another embodiment, the lipid blend comprises from about 15 mole% to about 29 mole% cholesterol relative to the lipid blend. In yet another embodiment, the lipid blend comprises from about 17.5 mol% to about 28 mol% cholesterol relative to the lipid blend.
In another embodiment, the particle-forming component is free of fatty acids or cationic lipids (i.e., lipids that carry a net positive charge at physiological pH).
In another embodiment, the particle-forming component comprises a hydrophilic polymer in which long chains of highly hydrated flexible neutral polymers are attached to phospholipid molecules. Without being bound by any theory, it is believed that the hydrophilic polymer stabilizes the liposomes and results in longer circulation times in vivo. Examples of hydrophilic polymers include, but are not limited to: polyethylene glycol (PEG), methoxy PEG (mPEG), ganglioside GM having a molecular weight of about 2,000 to about 5,000 daltons1Polysialic acid, polylactic acid (also known as polylactide), polyglycolic acid (also known as polyglycolide), polylactic polyglycolic acid (also known as polyglycolide), polyvinyl alcohol, polyvinylpyrrolidone, polymethyloxazoline (polymetaxazoline), polyethyloxazoline, polyhydroxyethyloxazoline, polyhydroxypropyloxazoline, polyasparagine, polyhydroxypropylmethacrylamide, polymethacrylamide, polydimethylacrylamide, polyvinylmethylether, polyhydroxyethylacrylate, derivatized celluloses (such as hydroxymethylcellulose or hydroxyethylcellulose), and synthetic polymers.
The particle-forming component may further comprise a lipid conjugate of an antibody or peptide that functions as a targeting moiety to enable the liposome to specifically bind to a target cell having a target molecule. Examples of target molecules include, but are not limited to: epidermal Growth Factor Receptor (EGFR), vascular endothelial growth factor receptor (VEGF), carcinoembryonic antigen (CEA), and erbB-2/neu (HER 2).
Liposomes for use in the arthritis treatment described herein can be produced by conventional techniques for preparing vesicles. These techniques include: ether injection (Deamer et al, Acad. Sci. (1978)308:250), surfactant (Brunner et al, Biochim. Biophys. acta (1976)455:322), freeze-thaw (Pick et al, Arch. Biochim. Biophys. 1981)212:186), reverse phase evaporation (Szoka et al, Biochim. Biophys. acta (1980)601:55971), ultrasonication (Huang et al, Biochemistry (1969)8:344), ethanol injection (Kremer et al, Biochemistry (1977)16:3932), extrusion (Hope et al, Biochemistry. acta (1985)812:5565), French press method (French press method) (BarenFEtt et al, BS 9, Levy et al, Biophys. 1979, Biophys. 4679, Biozoc. 4670, Biozon et al, Biozon. 4679, Biozon et al, Biophys. 4679, 1980). All references set forth above describe methods and conventional techniques for forming liposomal vesicles, and the description of these methods is incorporated herein by reference.
In exemplary embodiments, the therapeutic agent is encapsulated in a liposome-containing component of a carrier agent, wherein the component of a carrier agent comprises a substantial amount of an aqueous medium, substantially free of neutral lipids (such as triglycerides), a non-aqueous phase (oil phase), a water-oil emulsion, or other mixture containing a non-aqueous phase. The drug-carrying component comprising a large amount of aqueous medium provides longer therapeutic efficacy and prolonged release profile of the therapeutic agent in the joint. In contrast, therapeutic agents encapsulated in a component carrying the agent comprising a large amount of non-aqueous medium (e.g., soybean oil medium) have faster Release characteristics and shorter therapeutic Efficacy (Bias et al, Sustained-Release Dexamethane Palmitate-pharmaceuticals and Efficacy in Patients with Activated injected pharmaceutical inhalation investment 2001; 21(6): 429-436).
In certain embodiments, the lipid mixture comprises one or more lipids that are not pre-formed into liposomes. The lipid mixture may be prepared by dissolving the lipids in suitable organic solvents including, but not limited to, ethanol, methanol, t-butanol, diethyl ether and chloroform and drying by conventional methods of heating, vacuum evaporation, nitrogen evaporation, freeze drying or other solvent removal.
After sterilization, the lipid solution is mixed with the therapeutic agent and lyophilized to form a powder or cake. Generally, at least one cryoprotectant and at least one buffer are added to effectively lyophilize the steroid-lipid mixture.
Cryoprotectants include, but are not limited to: mannitol, glycerol, dextrose, sucrose and/or trehalose. One exemplary cryoprotectant is mannitol.
Buffers include, but are not limited to: sodium dihydrogen phosphate dihydrate and disodium hydrogen phosphate anhydrous.
Some examples of lipid mixture preparation are described below to exemplify the preparation method of the lipid mixture, as they relate to the present invention.
Therapeutic agents
The therapeutic agent can be a steroid solution, a non-steroidal anti-inflammatory drug (NSAID), such as indomethacin, a disease-modifying anti-rheumatic drug (DMARD), or a combination of two or more of the foregoing, as well as combinations of one or more of the foregoing with other ingredients or compounds not specifically listed herein. DMARDs include small molecule agents such as methotrexate, leflunomide, sulfasalazine, cyclophosphamide, azathioprine, cyclosporine A, d-penicillamine, antimalarials (e.g., hydroxychloroquine). DMARDs also include biological substances such as tumor necrosis factor a (TNF-a) antagonists (e.g., etanercept, trade name Enbrel, commercially available from Wyeth pharmaceuticals, Inc., Collegeville, USA; adalimumab, trade name HUMIRA, commercially available from Abbott Laboratories, Abbott Park, Illinois, USA), interleukin-1 receptor antagonists, interleukin-6 receptor antagonists, anti-CD 20 monoclonal antibodies, CTLA-4-Ig, RGD peptides, and the like.
In an exemplary embodiment, the therapeutic agent is a substantially water-soluble steroid solution, such as DSP. In another exemplary embodiment, the therapeutic agent is a substantially water-soluble NSAID, such as a pharmaceutically acceptable salt of indomethacin. In yet another exemplary embodiment, the therapeutic agent is a substantially water-soluble DMARD, such as a pharmaceutically acceptable salt of methotrexate or a TNF-a antagonist. In yet another exemplary embodiment, the therapeutic agent is not covalently bound to a phospholipid or a fatty acid, such as a palmitate.
One or more therapeutic agents may be combined with pharmaceutically acceptable excipients and other ingredients suitable for pharmaceutical formulations, including formulations for human and animal use, as well as formulations for research, experimental and related uses. In some embodiments, a citrate buffer, preferably sodium citrate, is used. In other embodiments, a chelating agent, preferably EDTA, is used.
Water soluble steroids include any naturally occurring steroid hormone, synthetic steroids and derivatives thereof. Water soluble steroids include, but are not limited to: cortisone, hydrocortisone, prednisolone, methylprednisolone, prednisone, Dexamethasone Sodium Phosphate (DSP), hydrocortisone-17-valerate, flucortisone, fludrocortisone, paramethasone, and eplerenone. In one example, but not by way of limitation, the water soluble steroid is DSP. For example, a DSP solution of about 2mg/mL to about 100mg/mL can be used to reconstitute the lipid mixture.
Pharmaceutically acceptable salts of water soluble steroids include non-toxic salts formed from non-toxic inorganic or organic bases. For example, non-toxic salts may be formed with inorganic bases such as alkali or alkaline earth metal (e.g., potassium, sodium, lithium, calcium, or magnesium) hydroxides, or with organic bases such as amines and the like.
Pharmaceutically acceptable salts of water soluble steroids also include non-toxic salts formed from non-toxic inorganic or organic acids. Examples of organic and inorganic acids are hydrochloric acid, sulfuric acid, phosphoric acid, acetic acid, succinic acid, citric acid, lactic acid, maleic acid, fumaric acid, palmitic acid, cholic acid, pamoic acid, mucic acid, D-glutamic acid, glutaric acid, glycolic acid, phthalic acid, tartaric acid, lauric acid, stearic acid, salicylic acid, sorbic acid, benzoic acid, and the like.
Sustained release composition
in some exemplary embodiments, the lipid mixture consists essentially of one or more phospholipids and cholesterol as lipid components, suitable exemplary aqueous solutions or aqueous media for reconstitution include, but are not limited to, buffers, distilled water, saline, sugar solutions (e.g., sucrose solutions), etc. in other embodiments, the lipid mixture consists essentially of one or more phospholipids, a cholesterol solution, and a phospholipid-phospholipid mixture, and optionally a phospholipid-phospholipid mixture, wherein the lipid mixture is in the form of a phospholipid-phospholipid mixture, and optionally a phospholipid-phospholipid mixture, wherein the lipid mixture is in the form of a phospholipid-phospholipid mixture, optionally a phospholipid-phospholipid mixture, and optionally a phospholipid-phospholipid mixture, wherein the phospholipid-phospholipid mixture is in the form of a phospholipid-phospholipid mixture, optionally a phospholipid-phospholipid mixture, and optionally a phospholipid-phospholipid mixture, optionally a phospholipid-and optionally a phospholipid mixture, optionally a phospholipid-protecting lipid-lipid mixture, and optionally a phospholipid mixture, optionally a phospholipid-phospholipid mixture, optionally a phospholipid-phospholipid mixture, and optionally a phospholipid-phospholipid mixture, optionally a phospholipid.
In some embodiments, the sustained release composition further comprises at least one pharmaceutically acceptable excipient, diluent, vehicle, carrier, vehicle for active ingredients, preservative, cryoprotectant, or a combination thereof.
In one embodiment, the sustained release composition of the invention is prepared by preparing a lipid mixture and reconstituting it with a therapeutic agent to form an aqueous suspension.
In another embodiment, the sustained release composition of the invention is prepared by adding the therapeutic agent to the lipid mixture during the preparation of the lipid mixture, followed by lyophilization of the combination of the lipid mixture and the therapeutic agent together with one or more cryoprotectants to form a powder.
In an exemplary embodiment, the sustained release composition comprises a water soluble steroid having a potency equivalent to a dose of about 2mg to about 8mg dexamethasone. For example, the efficacy of 4mg DSP in the sustained release composition is equivalent to the efficacy of 3mg dexamethasone. The efficacy of 10mg DSP in the sustained release composition was equivalent to that of 7.6mg dexamethasone. Similarly, the potency of 40mg methylprednisolone acetate is equivalent to that of 7.5mg dexamethasone.
The sustained release compositions described herein are useful for treating a subject suffering from arthritis (such as rheumatoid arthritis).
In one embodiment, from about 50% to about 95% of the therapeutic agent in the sustained release composition is in unassociated form (i.e., from about 5% to about 50% of the therapeutic agent is in associated form). In another embodiment, about 60% to 90% of the therapeutic agent in the sustained release composition is in unassociated form. The term "therapeutic agent in unassociated form" refers to a therapeutic molecule that is separable from the phospholipid/cholesterol portion of the sustained release composition via gel filtration.
In another embodiment, the weight ratio of combined phospholipids and cholesterol to therapeutic agent is from about (5-80) to about 1. In another embodiment, the weight ratio of combined phospholipids and cholesterol to therapeutic agent is (5-40): 1. For example, the weight ratio of combined phospholipid and cholesterol to therapeutic agent is about (5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, or 80) to about 1.
Method for treating arthritis
The present invention relates to a method of treating arthritis in an individual, comprising administering to an individual in need thereof an effective amount of a sustained release composition as described herein, thereby alleviating a symptom and/or sign of arthritis in said individual.
Sustained release compositions are formulated to be suitable for IA, intramuscular or subcutaneous administration. Intra-articular injection comprises the following steps: 1) confirming and marking the proper injection position of the joint to be treated; 2) sterilizing the injection site using sterile techniques and optionally providing a local anesthetic; 3) the needle is inserted into the joint space at the injection site. Needle insertion may optionally be performed under ultrasound guidance. A small amount of synovial fluid was aspirated to confirm that the needle tip was within the joint space. 4) The drug is injected into the joint space.
The dosage of the sustained-release composition of the present invention can be determined by one skilled in the art according to the embodiment. A unit dosage form or multiple dosage forms are contemplated, each of which provides advantages in certain clinical configurations. According to the present invention, the actual amount of the sustained-release composition administered may vary depending on the age, weight, condition, joint type of the individual to be treated and depends on the judgment of the medical professional.
The dose of DSP used for IA injection depends on the patient condition and joint size. In an exemplary embodiment, the dose of DPS is from about 0.2mg to about 6mg per IA injection. In another exemplary embodiment, for large joints (such as the knee joint), the dose of DPS is from about 2mg to about 4mg per IA injection. In yet another exemplary embodiment, for small joints, such as interphalangeal joints, the dose of DPS is from about 0.8mg to about 1mg per IA injection.
In an exemplary embodiment, the dose of indomethacin per IA injection is from about 5mg to about 30 mg. In another exemplary embodiment, the dose of indomethacin per IA injection is from about 10mg to about 25 mg. In yet another exemplary embodiment, the dose of indomethacin per IA injection is from about 15mg to about 20 mg.
In an exemplary embodiment, the dose of methotrexate per IA injection is from about 1mg to about 15 mg. In another exemplary embodiment, the dose of methotrexate per IA injection is from about 5mg to about 12.5 mg. In yet another exemplary embodiment, the dose of methotrexate per IA injection is from about 7.5mg to about 10 mg.
The frequency of IA injections varies daily, once every three to five days, once a week (weekly), or once every two to three weeks.
The following examples will serve to further illustrate the invention without, however, constituting any limitation thereto. On the contrary, it is to be clearly understood that resort may be had to various embodiments, modifications, and equivalents thereof which, after reading the description herein, may suggest themselves to those skilled in the art without departing from the spirit of the present invention. Unless otherwise indicated, during the studies described in the following examples, conventional procedures were followed. For illustrative purposes, some procedures are described below.
The publications cited herein and the materials to which they are cited are specifically incorporated by reference in their entirety. Various embodiments have been described. It will be understood that various changes may be made. Accordingly, other embodiments are within the scope of the following claims.