Patent Application
20140364371
The contents of the text file submitted electronically herewith are incorporated herein by reference in their entirety: A computer readable format copy of the Sequence Listing (filename: PHAS—004—01US_SeqList.txt, date recorded: Apr. 18, 2011, file size 47 kilobytes).
The present invention concerns methods and compositions for the controlled released delivery of pharmaceutical compounds.
Osteoarthritis (OA) is a degenerative joint disease that affects more patients than any other musculoskeletal disorder. It accounts for over 4 million hospitalizations every year and its prevalence is estimated to increase by over 50% in the next 20 years. OA affects individual joints and was historically considered to arise from a “wear and tear” due to joint loading. OA is now understood to arise from multiple causes, including biochemical, genetic and biomechanical factors, that interact and promote the progression of joint disease. While surgical options for joint re-alignment (osteotomy), prosthetic replacement (arthroplasty), or fusion (arthrodesis) are available to patients with advanced joint degeneration, only a small fraction of patients need operative treatments. Hence, there is a great and increasing need to provide non-surgical treatments that are capable of both alleviating patient symptoms and modifying the course of disease progression.
The most widely available treatments for the patient with early to moderate joint disease are the chronic use of NSAIDs. Although these drugs provide effectiveness in pain relief, they are associated with frequent adverse conditions including gastrointestinal bleeding and ulceration. Additionally. NSAIDs are reported to inhibit cell biosynthesis which may compromise the long-term intrinsic repair process in joint degeneration. More recently protein drugs with the ability to modify the disease state of OA have been identified. These protein drugs include TNF-a inhibitors and IL-1 inhibitors have shown significant promise both in preclinical and clinical studies in preventing the onset and retarding the progression of the disease.
Since OA is a disease that is localized to a few joints at a time, localized delivery of the therapeutic is highly preferred. One such technique is the administration of the drug directly into the affected joint cavity, i.e. intra-articular injection, which is currently recommended for corticosteroids and hyaluronan solutions in treating OA. Although the intra-articular mechanism of drug delivery is attractive to the patient and clinician alike, it is compromised by the presence of a highly efficient lymphatic system that rapidly clears molecules from the synovial cavity. Consequently, the therapeutic drug has to be administered frequently or at high concentrations to be effective. This, in turn, may be costly and result in adverse side effects and high levels of patient discomfort. Controlled drug delivery systems have been sought-after to overcome these challenges.
U.S. Pat. No. 6,328,996 to Urry describes bioelastomeric drug delivery systems, but does not suggest them for administration directly into a region of interest.
An objective of the present invention is to provide a thermally responsive biocompatible polymer for the sustained delivery of drags such as IL-1 receptor antagonist. The delivery system in the present invention utilizes the unique rapid aggregation and slow disaggregation properties of thermally responsive biopolymers to deliver protein drugs directly to a pathologic site, such as an OA affected joint.
The present invention provides a method for delivering a drug depot of a compound of interest to a selected region in a subject. The method comprises administering a composition directly to said region of interest, the composition comprising said compound to be delivered and a polymer that undergoes an inverse temperature phase transition, so that a sustained release of said compound of interest at said selected region is provided.
In some embodiments the present invention provides a method for delivering a drug depot of a compound of interest to a selected region in a subject (e.g., a joint or synovial joint). The method comprises administering a composition to region of interest (e.g., directly, such as by injection in or to the region of interest). The composition comprises the compound to be delivered and a polymer that undergoes an inverse temperature phase transition. The polymer has a transition temperature (Tt) less than the body temperature of the subject (e.g., less than 37° C.). The composition or conjugate aggregates in the region of interest and then gradually disaggregates, providing a sustained or controlled release of the compound of interest at the selected region.
In some embodiments the present invention provides a method for delivering a drug depot of a compound of interest to a selected region in a subject, said method comprising: administering a composition directly to said region of interest, said composition comprising said compound to be delivered and a polymer that undergoes an inverse temperature phase transition; wherein said polymer has a transition temperature (Tt) less than the body temperature of said subject: so that said composition (or said conjugate) separates from solution in said region of interest to form a bulk aggregate, and then gradually separates from bulk aggregate to go back to solution, providing a sustained release of said compound of interest at said selected region (or stated differently, so that said conjugate separates from solution in said region of interest and then gradually goes back to solution phase, providing a sustained release of said compound of interest at said selected region).
In some embodiments the composition comprises the compound to be delivered conjugated to the polymer; in other embodiments the composition comprises the compound to be delivered mixed with, but not otherwise conjugated to or chemically coupled to, the polymer.
A further aspect of the invention is a pharmaceutically acceptable composition comprising a therapeutic compound in combination with (e.g., mixed with or conjugated to) a polymer that undergoes an inverse temperature phase transition. Examples of such therapeutic compounds include but are not limited to TNF antibodies, IL-1 antibodies, soluble TNF receptors, soluble IL-1 receptors, TNF receptor antagonists, and IL-1 receptor antagonists, with a particular example being recombinant human IL-1 receptor antagonist and its isoforms.
A further aspect of the invention is an injection device (including syringes and other injection devices) containing a composition as described herein.
A further aspect of the invention is the use of an injection device as described herein for carrying out a method as described herein.
“Bioelastic polymer” as used herein refers to compounds that comprise repeating elastomeric units. The elastomeric units are typically pentapeptides, tetrapeptides, of nonapeptides. Examples are elastin-like polypeptides or “ELPs”.
“Subjects” as used herein includes human subjects (including both male and female subjects and including juvenile, adolescent, adult, and geriatric subjects), as well as animal subjects, particularly other mammalian subjects such as primates, dogs, cats, and horses, for veterinary purposes.
“Arthritis” as used herein means any type of arthritis, including but not limited to rheumatoid arthritis, osteoarthritis, and ankylosing spondylitis.
“Aggregate” as used herein means a collection or agglomeration of a plurality of molecules such that a particle is formed.
“Dis-aggregation” as used herein means a separation of molecule or molecules from an aggregate, a progressive diminishing of aggregate size; or a progressive diminishing of particle size.
“Region of interest” as used herein may be any region of interest, including but not limited to joints and solid tumors (“solid tumor” including the resection cavities within solid tumors after surgical removal of the primary mass thereof and into which the compositions of the invention may be directly administered). In some embodiments of joint administration, the region of interest is an intervetebral disk space or a related spinal joint structure.
“Body temperature” as used herein includes both core body temperature and regional body temperature (e.g., the temperature of an extremity when a region of interest such as a joint is located in an extremity).
“Joint” as used herein refers to a movable point where two bones meet, often a synovial joint or syndesmosis, and including but not limited to ball and socket joints, ellipsoidal joints, gliding joints, hinge joints, pivot joints and saddle joints. Such joints may be located in, for example, the shoulder, neck, spine, elbow, hip, wrist, hand, knee, ankle, or foot.
“Solid tumor” as used herein may be any solid tumor or cancer, including but not limited to primary and secondary (or metastatic) solid tumors of lung cancer, liver cancer, prostate cancer, ovarian cancer, colon cancer, skin cancer (including melanoma), brain cancer, etc.
“Injection” or “injecting” as used herein may be carried out by any suitable means through a needle, syringe, shunt, cannula (e.g., of 7-33 gauge) or any other suitable device that delivers the composition to be delivered directly into the region of interest (in contrast to and not including systemic delivery), as a single bolus or as an infusion over time.
“Antibody” or “antibodies” as used herein refers to all types of immunoglobulins, including IgG, IgM, IgA, IgD, and IgE. The term “immunoglobulin” includes the subtypes of these immunoglobulins, such as IgG1, IgG2, IgG3, IgG4, etc. Of these immunoglobulins, IgM and IgG are preferred, and IgG is particularly preferred. The antibodies may be of any species of origin, including (for example) mouse, rat, rabbit, horse, or human, or may be chimeric antibodies. The term “antibody” as used herein includes antibody fragments which retain the capability of binding to a target antigen, for example, Fab, F(ab′)2, and Fv fragments, and the corresponding fragments obtained from antibodies other than IgG. Such fragments are also produced by known techniques.
“Chemotherapeutic agent” (or “anticancer agent”) as used herein includes but is not limited to methotrexate, daunomycin, mitomycin, cisplatin, vincristine, epirubicin, fluorouracil, verapamil, cyclophosphamide, cytosine arabinoside, aminopterin, bleomycin, mitomycin C, democolcine, etoposide, mithramycin, chlorambucil, melphalan, daunorubicin, doxorubicin, tamosifen, paclitaxel, vincristin, vinblastine, camptothecin actinomycin D, and cytarabine.
The disclosures of all United States patent references cited herein are to be incorporated by reference herein in their entirety.
Compounds of interest. Any suitable compound, including but not limited to proteins and peptides, may be coupled to the polymer (e.g., via chemical bond or production as a fusion protein) or mixed with the polymer as noted above. In general, the compounds of interest may be detectable groups or a therapeutic group.
For example, the compound of interest may be selected from the group of bone morphogenetic proteins, peptides, and growth factors, more particularly human calcitonin analogs, osteogenic growth peptides, and osteogenic growth peptide-human calcitonin analog hybrids. See, e,g., U.S. Pat. No. 6,593,394.
The compound of interest may be selected from the group of antiinfectives such as antibiotics and antiviral agents; chemotherapeutic agents (i.e. anticancer agents); anti-rejection agents; analgesics and analgesic combinations; anti-inflammatory agents; hormones such as steroids; growth factors, including bone morphogenic proteins (i.e. BMP's 1-7), bone morphogenic-like proteins (i.e. GDF-5, GDF-7 and GDF-8), epidermal growth factor (EGF), fibroblast growth factor (i.e. FGF 1-9), platelet derived growth factor (PDGF), insulin like growth factor (IGF-I and IGF-II), transforming growth factors (i.e. TGF-.beta. I-III), vascular endothelial growth factor (VEGF); and other naturally derived or genetically engineered proteins, polysaccharides, glycoproteins, or lipoproteins. See, e.g., D. Overaker, U.S. Pat. No. 6,575,986.
The compound of interest may be compounds or agents that actually promote or expedite healing, the effectors may also include compounds or agents that prevent infection (e.g., antimicrobial agents and antibiotics), compounds or agents that reduce inflammation (e.g., anti-inflammatory agents), compounds that prevent or minimize adhesion formation, such as oxidized regenerated cellulose (e.g., INTERCEED™ and SURGICEL™, available from Ethicon, Inc.), hyaluronic acid, and compounds or agents that suppress the immune system (e.g., immunosuppressants). Suitable compounds of interest include heterologous or autologous growth factors, proteins (including matrix proteins), peptides, antibodies, enzymes, platelets, glycoproteins, hormones, cytokines, glycosaminoglycans, nucleic acids, analgesics, viruses, virus particles, and cell types. It is understood that one or more effectors of the same or different functionality may be incorporated within the scaffold. Suitable compounds of interest include the multitude of heterologous or autologous growth factors known to promote healing and/or regeneration of injured or damaged tissue. Suitable compounds of interest include chemotactic agents; therapeutic agents (e.g., antibiotics, steroidal and non-steroidal analgesics and anti-inflammatories, anti-rejection agents such as immunosuppressants and anti-cancer drugs); various proteins (e.g., short term peptides, bone morphogenic proteins, glycoprotein and lipoprotein); cell attachment mediators; biologically active ligands; integrin binding sequence; ligands; various growth and/or differentiation agents and fragments thereof (e.g., epidermal growth factor (EGF), hepatocyte growth factor (HGF). IGF-I, IGF-II, TGF-.beta. I-III, growth and differentiation factors, vascular endothelial growth factors (VEGF), fibroblast growth factors (FGF), platelet derived growth factors (PDGF), insulin derived growth factor (IGF) and transforming growth factors, parathyroid hormone, parathyroid hormone related peptide, bFGF; TGFB superfamily factors; BMP-2; BMP-4; BMP-6; BMP-12; sonic hedgehog; GDF5; GDF6; GDF8; MP52, CDMP1); small molecules that affect the upregulation of specific growth factors; tenascin-C; hyaluronic acid; chondroitin sulfate; fibronectin; decorin; thromboelastin; thrombin-derived peptides; heparin-binding domains; heparin; heparan sulfate; DNA fragments and DNA plasmids. Suitable effectors likewise include the agonists and antagonists of the agents noted above. The growth factor can also include combinations of the growth factors listed above. In addition, the growth factor can be autologous growth factor that is supplied by platelets in the blood. In this case, the growth factor from platelets will be an undefined cocktail of various growth factors. See, e.g., J. Hwang et al., US Patent Application Publication No. 2004/0267362.
In some embodiments, particularly for the treatment of arthritis, the compound of interest is an antiinflammatory agent, examples of which include but are not limited to TNF antibodies, IL-1 antibodies, soluble TNF receptors, soluble IL-1 receptors, TNF receptor antagonists, IL-1 receptor antagonists, COX-2 inhibitors, and non-steroidal antiinflammatory agents (including active fragments thereof for protein or peptide compounds such as antibodies and receptors). Numerous examples of such compounds are known. See, e.g., U.S. Pat. No. 6,846,834; see also U.S. Pat. Nos. 6,624,184; 6,596,746; 6,498,165; and 6,420,373. One particular example is rhIL1Ra (including its isoforms) (commercially available as KINERET™ from Amgen), as described in U.S. Pat. Nos. 6,599,873 and 5,075,222.
Bioelastic polymers. Bioelastic polymers are known and described in, for example, U.S. Pat. No. 5,520,672 to Urry et al. In general, bioelastic polymers are polypeptides comprising elastomeric units of bioelastic pentapeptides, tetrapeptides, and/or nonapeptides. Thus in some embodiments the elastomeric unit is a pentapeptide, in other embodiments the elastomeric unit is a tetrapeptide, and in still other embodiments the elastomeric unit is a nonapeptide. Bioelastic polymers that may be used to carry out the present invention are set forth in U.S. Pat. No. 4,474,851, which describes a number of tetrapeptide and pentapeptide repeating units that can be used to form a bioelastic polymer. Specific bioelastic polymers that can be used to carry out the present invention are also described in U.S. Pat. Nos. 4,132,746; 4,187,852; 4,500,700; 4,589,882; and 4,870,055. Still other examples of bioelastic polymers are set forth in U.S. Pat. No. 6,699,294 to Urry, U.S. Pat. No. 6,753,311 to Fertala and Ko, U.S. Pat. No. 6,063,061 to Wallace and U.S. Pat. No. 6,852,834 to Chilkoti. The bioelastic polymers may contain additional residues or units such as leader and/or trailer sequences as is known in the art.
In one nonlimiting example, the bioelastic polymers used to carry out the present invention are polypeptides that contain at least the general block structure, [(VPGXiG)ni]m (SEQ ID NO:4) for i=1 to J, Xi is any amino acid other than proline (e.g., Ala, Leu, Phe), ni is the number of successive repeats of block i adjacent to and preceding block i+1. ni is any number such as 1, 2, 3, or 4 up to 20 and ni are independent of i. J is any number such as 1, 2, or 3 to 5 or more. m is the number of repeats of the block structure where m is any suitable number such as 2, 3 or 4 up to 60, 80 or 300 or more. The identity and frequency of the various amino acids at the fourth position can be changed. For example, the bioelastic polymers used to carry out the present invention may be polypeptides of the general formula: [(VPGAG)51(VPGVG)2(VPGGG)23]m, (SEQ ID NO:5) where X1=A, X2=V, X3=G, n1 is 5, n2 is 1, n3 is 2, m is at least 1, 2, or 3 up to 100, 150 or 300 or more.
In another nonlimiting example, bioelastic polymers used to carry out the present invention may comprise repeating elastomeric units selected from the group consisting of bioelastic nonpeptides, pentapeptides and tetrapeptides, where the repeating units comprise amino acid residues selected from the group consisting of all amino acid and glycine residues. Preferred amino acid residues are selected from the group consisting of alanine, valine, leucine, isoleucine, proline, phenylalanine, tryptophan, and methionine. In many cases, the first amino acid residue of the repeating unit is a residue of valine, leucine, isoleucine or phenylalanine; the second amino acid residue is a residue of proline; the third amino acid residue is a residue of glycine; and the fourth amino acid residue is glycine or a very hydrophobic residue such as tryptophan, phenylalanine or tyrosine. Particular examples include the tetrapeptide Val-Pro-Gly-Gly (SEQ ID NO:6), the tetrapeptide GGVP (SEQ ID NO:7), the tetrapeptide GGFP (SEQ ID NO:8), the tetrapeptide GGAP (SEQ ID NO:9), the pentapeptide is Val-Pro-Gly-Val-Gly (SEQ ID NO:10), the pentapeptide GVGVP (SEQ ID NO:11), the pentapeptide GKGVP (SEQ ID NO:12), the pentapeptide GVGFP (SEQ ID NO:13), the pentapeptide GFGFP (SEQ ID NO:14), the pentapeptide GEGVP (SEQ ID NO:15), the pentapeptide GFGVP (SEQ ID NO:16), and the pentapeptide GVGIP (SEQ ID NO:17). See, e.g., U.S. Pat. No. 6,699,294 to Urry.
Administration. As noted above, a method of the invention generally comprises administering a composition to region of interest (e.g., directly, such as by injection in or to the region of interest). The composition comprises the compound to be delivered and a polymer that undergoes an inverse temperature phase transition. The polymer has a transition temperature (Tt) less than the body temperature of the subject (e.g., less than 37° C.). The conjugate aggregates in the region of interest and then gradually disaggregates, providing a sustained release of the compound of interest at the selected region. The administering step may be carried out by any suitable means, such as injection, and particularly intra-articular injection (e.g., into the synovial fluid of a synovial joint).
Patients afflicted with any disease or condition for which a depot drug is desired can be treated by the methods, with a particular example being patients afflicted with arthritis.
The compound of interest can be administered in any suitable amount depending upon the site of injection, age, weight, and condition of the subject, particular active agent, etc. In general the compound is administered in an amount of from 0.001, 0.01, 0.1, 1, 5 or 10 milligrams per administration or injection, up to about 100, 200 or 300 milligrams per administration or injection. In general the compound is administered at a concentration of 1 pg/ml to 500 mg/ml, with the injection volume dependent upon the region of interest. In general the ELP ma also be co-administered at an equivalent concentration of 1 pg/ml to 500 mg/ml, or much higher concentrations in the example of mixing compound with elastomer. In some embodiments the compound of interest preferably has an in vivo half life in said region of one or two hours or more; in some embodiments administration of the compound of interest in combination with ELP in the said administration method is associated with an in vivo half life for the compound in said region of at least 2 or 3 days, one week, or four weeks or more, up to 2 or three months or more. Stated otherwise, it is contemplated that the administering step will be carried out on a regular and repeated basis (e.g., for the treatment of a chronic condition such as arthritis), but that the frequency of administration will not be greater than, one, two, three or four times per month, while still retaining the desired treatment-effective amount.
The present invention is explained in greater detail in the following non-limiting Examples.
Elastin like polypeptides (ELPs) are genetically engineered biopolymers, made up of the pentapeptide repeat unit as defined above that undergo aggregation, in response to an increase in temperature. ELPs are soluble at temperatures below their transition temperature (Tt), and become insoluble and form micron size aggregates at temperatures above their Tt. We disclose herein that a protein drug attached to this drug carrier will aggregate at the time of injection and form a drug ‘depot’ at the injection site, which will then slowly disaggregate into the joint space in a sustained manner (see, e.g.,
Elastin-like polypeptides (ELPs) or bioelastic polymers were evaluated for an ability to provide sustained release of “free” polymer from aggregate form as a proof-of-concept of the proposed delivery system for the treatment of localized diseases, such as osteoarthritis. The drug delivery system utilizes the unique ELP properties of rapid thermally-induced aggregation to form large particles, followed by sustained release of the ELP from the bulk aggregated large particles, termed a “free” form. To evaluate this, two ELP molecules were synthesized utilizing genetic engineering techniques and thermally purified: (1) ELP4-90 [(VPGVG)10]9 (SEQ ID NO:1), Tt=30.6° C., MW=37 kDa; and (2) ELP4-120=[(VPGVG)10]12 (SEQ ID NO:2). Tt=28.6° C. MW=49 kDa, in accordance to known techniques (see U.S. Pat. No. 6,699,294 to Urry and U.S. Pat. No. 6,852,834 to Chilkoti). For the in vitro study, 1.5 ml of either ELP at a concentration of 30 mg/ml was placed in conical tubes and incubated at 37° C. for 24 h. At the end of this time, the supernatant was completely exchanged for PBS at 37° C. and the quantity of ELP in the supernatant quantified as a function of starting ELP (
For both ELP types, the polypeptide underwent an inverse temperature phase transition shortly after being placed at 37° C. and slowly reached equilibrium with 15-20% of the starting ELP in a free form (
The biodistribution and joint half-life of an aggregating ELP4-120 ([(VPGVG)10]12, SEQ ID NO:2, Tt<32° C., MW 47 kDa) and non-aggregating, ELP2 ([(VPGVG)1(VPGAG)8(VPGGG)7]10, SEQ ID NO:18, MW 61 kDa, Tt>50° C.). ELPs following intra-articular injection were evaluated in a rat animal model. The ELPs were labeled with [14C] to yield a specific activity between 32-37 mCi/mmole. The labeled ELF was dialyzed, sterile filtered (0.22 μm filter) and a volume of 30 μl at ˜650 μM of either peptide was injected into the joint cavity of the right knee of Wistar rats. After injection, the animals were housed in a cage for up to 4 weeks. At each time point five rats were sacrificed, their right and left knee (synovial fluid, meniscus, cartilage, synovium), blood, heart, lung, liver, spleen, kidney, and bladder were collected and digested. The radioactivity of each tissue was determined using liquid beta-scintillation counting. Total counts per tissue weight were measured for each animal and for each tissue or body fluid (n=5 animals per group and per time point) and plotted for each tissue as a function of time. Values for joint tissues and joint fluids were summed into a total “joint” value (synovial fluid, meniscus, joint cartilages, synovium).
The total radioactivity recovered from all tissues and fluids in the joint cavity at 10 min after injection was adjusted for injected [14C]-ELP that was non-specifically distributed, with the starting dose found to be between 70-75% of administered dose. Radioactivity per tissue weight was then normalized by this initial injected knee joint cavity value (10 min post-injection), in order to obtain the percent of injected dose per gram (% ID/gm) of tissue relative to the delivered amount for all recovered tissues and fluids.
At all times, no preferential accumulation of either peptide in the peripheral organs was observed (
A radioactive half-life (t1/2) was determined for the injected knee joint compartment by summing the total radioactivity count for the joint as described (
In proof-of-concept tests of this drug delivery system, two aggregating fusion proteins of ELP and IL1Ra were genetically synthesized as follows. The gene for human IL1Ra was PCR-cloned from LPS activated U937 cells as previously described by Carter et al. (1990) Nature 344: 633-8. Two ELP formulations were synthesized as described by U.S. Pat. No. 6,852,834 to Chilkoti: ELP1 [(VPGVG)5(VPGGG)3(VPGAG)2]9, (SEQ ID NO:20), MW 36 kDa, and ELP4 [(VPGVG)10]3, (SEQ ID NO:21), MW 14.5 kDa. Each ELP gene was then sub-cloned into the IL1Ra containing vector (pET-25b+) using traditional molecular biology techniques to produce two ELP-IL1Ra fusion proteins (see Tables 1-2) with molecular weights of 30.2 and 53 kDa. The fusion proteins were then expressed in E. coli and thermally purified in accordance with known technique U.S. Pat. No. 6,699,294 to Urry and U.S. Pat. No. 6,852,834 to Chilkoti. The size and purity of the fusion proteins was examined by SDS-PAGE and shown in
The temperature driven aggregation of each fusion protein was evaluated by dynamic light scattering, where the scattering of laser light by the fusion protein was monitored while a dilute solution (25 μM) of the fusion protein was heated at 1° C. interval over the range of 20-60° C. From the scatter data the hydrodynamic radius (R1t) was determined by the Stokes-Einstein relationship for spherical particles. The aggregation temperature was defined as the temperature above which large (>100 nm) particles start to form and the fusion protein aggregates.
The bioactivity of a represententative fusion protein (ELP4-IL1Ra) was examined for its ability to inhibit IL-1β augmented proliferation of Con A-stimulated murine C57 primary thymocytes as previously described. Briefly, primary thymocytes were mechanically isolated from freshly sacrificed 5 to 7 weeks old C57 mice and suspended in RPMI 1640 supplemented with 5% FCS, 1 μg/ml ConA (Sigma) and 1 ng/ml mIL-1β (Pierce) at a density of ˜2×106 cells/200 μl. The cells were then incubated in the presence of serial dilutions of either commercial rhIL1Ra (R&D Systems) or ELP4-IL1Ra fusion protein (as described above) for 48 hours. The proliferation of thymocytes was assessed by the uptake of [3H]-thymidine (0.5 μCi/well). Data were expressed as mean CPM±SD of [3H]-thymidine uptake of six replicate cultures. The highest concentration producing 50% inhibition (IC50) was determined by fitting the data to a sigmoid function. The ELP4-IL1Ra fusion protein showed significant bioactivity with an IC50 of 61.4±19.8 nM to inhibit 52 pM rmIL-1β, see
Examples of administration of compositions of the invention are given in
The foregoing is illustrative of the present invention, and is not to be construed as limiting thereof. The invention is defined by the following claims, with equivalents of the claims to be included therein.
This application is a continuation of application Ser. No. 11/472,113, filed Jun. 21, 2006, which claims the benefit of U.S. Provisional Application Ser. No. 60/693,966, filed Jun. 24, 2005, the disclosures of which are hereby incorporated by reference in their entirety.
This invention was made with Government support under Grant Nos. AR047442 and EB002263 from the National Institutes of Health. The Government has certain rights to this invention.
| Number | Date | Country | |
|---|---|---|---|
| 60693966 | Jun 2005 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 13012475 | Jan 2011 | US |
| Child | 14297531 | US | |
| Parent | 11472113 | Jun 2006 | US |
| Child | 13012475 | US |
