Patent Grant
9192648
The present application is a National Stage Application claiming the priority of co-pending PCT Application No. PCT/GB2006/003265 filed Sep. 5, 2006, which in turn, claims priority from Great Britain Application Serial No. 0518443.7, filed Sep. 9, 2005. Applicants claim the benefits of 35 U.S.C. §120 as to the PCT application and priority under 35 U.S.C. §119 as to the said Australian application, and the entire disclosures of both applications are incorporated herein by reference in their entireties.
The present invention relates to the use of agents that bind the complement protein C5 in the treatment of diseases associated with inappropriate complement activation, and in particular in the treatment of myasthenia gravis.
All documents mentioned in the text and listed at the end of this description are incorporated herein by reference.
The complement system is an essential part of the body's natural defence mechanism against foreign invasion and is also involved in the inflammatory process. More than 30 proteins in serum and at the cell surface are involved in complement system function and regulation. Recently it has become apparent that, as well as the ˜35 known components of the complement system which may be associated with both beneficial and pathological processes, the complement system itself interacts with at least 85 biological pathways with functions as diverse as angiogenesis, platelet activation, glucose metabolism and spermatogenesis (Mastellos, D., et al., Clin Immunol, 2005. 115(3): p. 225-35).
The complement system is activated by the presence of foreign antigens. Three activation pathways exist: (1) the classical pathway which is activated by IgM and IgG complexes or by recognition of carbohydrates; (2) the alternative pathway which is activated by non-self surfaces (lacking specific regulatory molecules) and by bacterial endotoxins; and (3) the lectin pathway which is activated by binding of manna-binding lectin (MBL) to mannose residues on the surface of a pathogen. The three pathways comprise parallel cascades of events that result in the production of complement activation through the formation of similar C3 and C5 convertases on cell surfaces resulting in the release of acute mediators of inflammation (C3a and C5a) and formation of the membrane attack complex (MAC). The parallel cascades involved in the classical and alternative pathways are shown in
Complement can be activated inappropriately under certain circumstances leading to undesirable local tissue destruction. Inappropriate complement activation has been shown to play a role in a wide variety of diseases and disorders including acute pancreatitis, Alzheimer's disease, allergic encephalomyelitis, allotransplatation, asthma, adult respiratory distress syndrome, burn injuries, Crohn's disease, glomerulonephritis, haemolytic anaemia, haemodialysis, hereditary angioedema, ischaemia reperfusion injuries, multiple system organ failure, multiple sclerosis, myasthenia gravis, ischemic stroke, myocardial infarction, psoriasis, rheumatoid arthritis, septic shock, systemic lupus erythematosus, stroke, vascular leak syndrome, transplantation rejection and inappropriate immune response in cardiopulmonary bypass operations. Inappropriate activation of the complement system has thus been a target for therapeutic intervention for many years and numerous complement inhibitors targeting different parts of the complement cascade are under development for therapeutic use.
In ischemic stroke and myocardial infarction, the body recognises the dead tissue in the brain or heart as foreign and activates complement so causing further local damage. Similarly in cardiopulmonary bypass operations, the body recognises the plastic surfaces in the machine as foreign, activates complement and can result in vascular damage. In autoimmune diseases, the body may wrongly recognise itself as foreign and activate complement with local tissue damage (e.g. joint destruction in rheumatoid arthritis and muscle weakness in myasthenia gravis).
Myasthenia gravis is a chronic autoimmune disease that results in progressive fatigue, loss of muscle tone and increasing paralysis. These symptoms are caused by inappropriate activation of complement resulting in an immune response directed against the nicotinic acetylcholine receptor (AchR) which leads, in turn, to reduced neuromuscular transmission. Myasthenia gravis may occur in association with other diseases such as a thymic tumor or thyrotoxicosis, as well as with rheumatoid arthritis and lupus erythematosus.
There is currently no cure for myasthenia gravis. The disease is usually treated initially using anticholinesterase agents, such as neostigmine bromide (Prostigmin) and pyridostigmine bromide (Mestinon), which help improve neuromuscular transmission and increase muscle strength. Treatment with anticholinesterase agents is, however, associated with adverse side effects caused from acetylcholine accumulation including gastrointestinal complaints and increased bronchial and oral secretions. In addition, although anticholinesterase agents often provide symptomatic benefit, they do not influence the course of the disease. Patients who do not respond to anticholinerterase agents may also be treated with long-term immunosuppressive drugs such as the cortocosteroid prednisone, or other immunosuppressant drugs such as cyclosporine, azathioprine and cyclophosphamide. These immunosuppressant drugs are, however, associated with serious side effect. Corticosteroids side effects include weight gain, osteoporosis, hypertension and glaucoma. Azathioprine and cyclosporine are associated with liver dysfunction and an increased risk of malignancy. In some cases, thymectomy is recommended as an alternative to drugs but the response is unpredictable and symptoms of the disease may continue for months or years after surgery.
Experimental autoimmune myasthenia gravis (EAMG) may be induced in animal models by immunisation with purified AChR or anti-AChR antibodies and these models are useful in assessing the effect of complement inhibitors on the progression of the disease. The complement inhibitor soluble complement receptor 1 (sCR1) has been shown to delay weight loss and reduce clinical signs of EAMG, suggesting that this molecule may be useful in the treatment for myasthenia gravis (Piddlesden et al, J. Neuroimmunol., 1996, 71: 173-177). However, daily injections of sCR1 were required to achieve these effects and, although sCR1 reduced weight loss, it did not completely prevent it. sCR1 does not therefore completely prevent the symptoms of myasthenia gravis.
Furthermore, sCR1 acts by binding early products of the complement cascade, C3b and C4b. The complement system plays an important and valuable role in defence against pathogens and many of the early by-products of the cascade are important in the recognition and opsonisation of pathogenic organisms. For this reason, therapeutic intervention in the earlier stages of the classical and alternative pathways is considered to carry the risk of increased susceptibility to microbial infection (Roos, A., et al., Immunobiology, 2002, 205(4-5): p. 595-609).
There is thus a great need for agents that improve upon the currently available treatments for myasthenia gravis.
Accordingly, the invention provides a method of treating or preventing myasthenia gravis comprising administering to a subject in need thereof a therapeutically or prophylactically effective amount of an agent that binds complement C5.
The invention also provides the use of a therapeutically or prophylactically effective amount of an agent that binds complement C5 in the manufacture of a medicament for treating or preventing myasthenia gravis.
Preferably, the agent acts to prevent the cleavage of complement C5 by C5 convertase into complement C5a and complement C5b-9.
The complement C5 protein, also referred to herein as C5, is cleaved by the C5 convertase enzyme, itself formed from C3a, an earlier product of the alternative pathway (
MAC is associated with other important pathological processes including rheumatoid arthritis (Neumann, E., et al., Arthritis Rheum, 2002. 46(4): p. 934-45; Williams, A. S., et al., Arthritis Rheum, 2004, 50(9): p. 3035-44), proliferative glomerulonephritis (Quigg, R. J., Curr Dir Autoimmun, 2004. 7: p. 165-80, idiopathic membranous nephropathy (Papagianni, A. A., et al., Nephrol Dial Transplant, 2002, 17(1): p. 57-63), proteinurea (He, C., et al., J Immunol, 2005. 174(9): p. 5750-7), demyelination after acute axonal injury (Mead, R. J., et al., J Immunol, 2002. 168(1): p. 458-65) and is also responsible for acute graft rejection following xenotransplantation (Nakashima, S., et al., J Immunol, 2002. 169(8): p. 4620-7).
C5a has become a target of particular interest in the field of complement-associated disorders (Mizuno, M. and D. S. Cole, Expert Opin Investig Drugs, 2005. 14(7): p. 807-21). Although C5a has many well-recognised pathological associations, the effects of its depletion in humans appear to be limited. Monoclonal antibodies and small molecules that bind and inhibit C5a or C5a receptors have been developed to treat various autoimmune diseases. These molecules do not, however, prevent the release of MAC.
In contrast, administration of an agent that binds C5 according to the first aspect of the invention, inhibits both the C5a peptide and the MAC. Surprisingly, it has been found that inhibition of both C5a and the MAC completely attenuates clinical symptoms associated with myasthenia gravis. Furthermore, because C5 is a late product of the classical and alternative complement pathways, inhibition of C5 is less likely to be associated with risks of concomitant infection that exist when targeting earlier products in the cascade (Allegretti, M., et al., Curr Med Chem, 2005. 12(2): p. 217-36).
The ability of an agent to bind C5 may be determined by standard in vitro assays known in the art, for example by western blotting following incubation of the protein on the gel with labelled C5. Preferably, the agent according to the invention binds C5 with an IC50 of less than 0.2 mg/ml, preferably less than 0.1 mg/ml, preferably less than 0.05 mg/ml, preferably less than 0.04 mg/ml, preferably less than 0.03 mg/ml, preferably 0.02 mg/ml, preferably less than 1 μg/ml, preferably less than 100 ng/ml, preferably less than 10 ng/ml, more preferably still, less than 1 ng/ml.
Preferably, the agent that binds C5 is derived from a haematophagous arthropod. The term “haematophagous arthropod” includes all arthropods that take a blood meal from a suitable host, such as insects, ticks, lice, fleas and mites. Preferably, the agent is derived from a tick, preferably from the tick Ornithodoros moubata.
According to one embodiment of the invention, the agent that binds C5 is a protein comprising amino acids 19 to 168 of the amino acid sequence in
According to an alternative embodiment, the protein used according to this embodiment of the invention may comprise or consist of amino acids 1 to 168 of the amino acid sequence in
The protein having the amino acid sequence given in
The EV576 protein and the ability of this protein to inhibit complement activation has been disclosed in WO2004/106369, where the EV576 protein was referred to as the “OmCI protein”. It has now been found that the EV576 protein is surprisingly effective in the treatment and prevention of myasthenia gravis. The data presented herein demonstrate that a single injection of EV576 totally attenuates weight loss and muscular weakness in the EAMG in mice for at least 7 days. EV576 is thus more effective in the treatment and prevention of EAMG than sCR1 which, as discussed above, only reduced the clinical symptoms of myasthenia gravis when administered on a daily basis (Piddlesden et al, J. Neuroimmunol., 1996, 71: 173-177). The surprising effectiveness of EV576 in the treatment of myasthenia gravis appears to be due to the fact that it acts by binding C5, thus inhibiting the activity of C5a and MAC. In addition, the data presented herein demonstrate that rEV576 is effective in models of both early mild myasthenia gravis and severe late stage myasthenic crises.
According to a further embodiment of the invention, the agent may be a nucleic acid molecule encoding the EV576 protein or a functional equivalent thereof. For example, gene therapy may be employed to effect the endogenous production of the EV576 protein by the relevant cells in the subject, either in vivo or ex vivo. Another approach is the administration of “naked DNA” in which the therapeutic gene is directly injected into the bloodstream or into muscle tissue.
Preferably, such a nucleic acid molecule comprises or consists of bases 53 to 507 of the nucleotide sequence in
The EV576 protein has been demonstrated to bind to C5 and prevent its cleavage by C5 convertase in rat, mouse and human serum with an IC50 of approximately 0.02 mg/ml. Preferably, functional equivalents of the EV576 protein which retain the ability to bind C5 with an IC50 of less than 0.2 mg/ml, preferably less than 0.1 mg/ml, preferably less than 0.05 mg/ml, preferably less than 0.02 mg/ml, preferably less than 1 μg/ml, preferably less than 100 ng/ml, preferably less than 10 ng/ml, more preferably still, less than 1 ng/ml.
The serum beta half-life of 125I labelled EV576 in rats has been found to be 30-38 hours. Preferably, the EV576 protein and its functional equivalents retain a half-life of greater than 20 hours, preferably greater than 25 hours, preferably greater than 30 hours, preferably greater than 40 hours, preferably greater than 50 hours, preferably greater than 100 hours.
In one respect, the term “functional equivalent” is used herein to describe homologues and fragments of the EV576 protein which retain its ability to bind C5, and to prevent the cleavage of complement C5 by C5 convertase into complement C5a and complement C5b-9. The term “functional equivalent” also refers to molecules that are structurally similar to the EV576 protein or that contain similar or identical tertiary structure, particularly in the environment of the active site or active sites of the EV576 protein that binds to C5, such as synthetic molecules.
The term “homologue” is meant to include reference to paralogues and orthologues of the EV576 sequence that is explicitly identified in
Methods for the identification of homologues of the EV576 sequence given in
Typically, greater than 30% identity between two polypeptides (preferably, over a specified region such as the active site) is considered to be an indication of functional equivalence and thus an indication that two proteins are homologous. Preferably, proteins that are homologues have a degree of sequence identity with the EV576 protein sequence identified in
Homologues of the EV576 protein sequence given in
Fragments of the EV576 protein and of homologues of the EV576 protein are also embraced by the term “functional equivalents” providing that such fragments retain the ability to bind C5. Fragments may include, for example, polypeptides derived from the EV576 protein sequence which are less than 150 amino acids, less than 125 amino acids, less than 100 amino acids, less than 75 amino acids, less than 50 amino acids, or even 25 amino acids or less, provided that these fragments retain the ability to bind to complement C5. Included as such fragments are not only fragments of the O. moubata EV576 protein that is explicitly identified herein in
A functional equivalent used according to the invention may be a fusion protein, obtained, for example, by cloning a polynucleotide encoding the EV576 protein in frame to the coding sequences for a heterologous protein sequence. The term “heterologous”, when used herein, is intended to designate any polypeptide other than the EV576 protein or its functional equivalent. Example of heterologous sequences, that can be comprised in the soluble fusion proteins either at N- or at C-terminus, are the following: extracellular domains of membrane-bound protein, immunoglobulin constant regions (Fc region), multimerization domains, domains of extracellular proteins, signal sequences, export sequences, or sequences allowing purification by affinity chromatography. Many of these heterologous sequences are commercially available in expression plasmids since these sequences are commonly included in the fusion proteins in order to provide additional properties without significantly impairing the specific biological activity of the protein fused to them (Terpe K, Appl Microbiol Biotechnol, 60: 523-33, 2003). Examples of such additional properties are a longer lasting half-life in body fluids, the extracellular localization, or an easier purification procedure as allowed by a tag such as a histidine or HA tag.
The EV576 protein and functional equivalents thereof, may be prepared in recombinant form by expression in a host cell. Such expression methods are well known to those of skill in the art and are described in detail by Sambrook et al (2000) and Fernandez & Hoeffler (1998). Recombinant forms of the EV576 protein and functional equivalents thereof are preferably unglycosylated.
The proteins and fragments of the present invention can also be prepared using conventional techniques of protein chemistry. For example, protein fragments may be prepared by chemical synthesis. Methods for the generation of fusion proteins are standard in the art and will be known to the skilled reader. For example, most general molecular biology, microbiology recombinant DNA technology and immunological techniques can be found in Sambrook et al. (2000) or Ausubel et al. (1991).
The subject to Which the agent that binds C5 is administered in the method or use of the invention is preferably a mammal, preferably a human. The subject to which the agent that binds C5 is administered may also be suffering from a further disease with which myasthenia gravis is associated, such as a thymic tumor, thyrotoxicosis, rheumatoid arthritis and lupus erythematosus.
The agent is administered in a therapeutically or prophylactically effective amount. The term “therapeutically effective amount” refers to the amount of agent needed to treat or ameliorate a targeted disease. The term “prophylactically effective amount” used herein refers to the amount of agent needed to prevent a targeted disease.
Preferably, the dose of the agent is sufficient to bind as much available C5 as possible in the subject, more preferably, all available C5. Preferably, the dose of the agent supplied is at least twice the molar dose needed to bind all available C5 in the subject. The dose of the agent supplied may be 2.5 times, 3 times or 4 times the molar dose needed to bind all available C5 in the subject. In one embodiment, the dose is from 1 mg/kg to 15 mg/kg. Preferably, the dose is from 1 mg/kg (mass of drug compared to mass of patient) to 10 mg/kg, preferably 2 mg/kg to 8 mg/kg.
The frequency with which the dose needs to be administered will depend on the half-life of the agent involved. Where the agent is the EV576 protein or a functional equivalent thereof, the dose may be administered on a daily basis, a twice daily basis, or every two, three, four days, five, six, seven, 10, 15 or 20 days or more.
The exact dosage and the frequency of doses may also be dependent on the patient's status at the time of administration. Factors that may be taken into consideration when determining dosage include the severity of the disease state in the patient, the general health of the patient, the age, weight, gender, diet, time and frequency of administration, drug combinations, reaction sensitivities and the patient's tolerance or response to therapy. The precise amount can be determined by routine experimentation, but may ultimately lie with the judgement of the clinician.
The agent will generally be administered as part of a pharmaceutically acceptable carrier. The term “pharmaceutically acceptable carrier”, as used herein, includes genes, polypeptides, antibodies, liposomes, polysaccharides, polylactic acids, polyglycolic acids and inactive virus particles or indeed any other agent provided that the carrier does not itself induce toxicity effects or cause the production of antibodies that are harmful to the individual receiving the pharmaceutical composition. Pharmaceutically acceptable carriers may additionally contain liquids such as water, saline, glycerol, ethanol or auxiliary substances such as wetting or emulsifying agents, pH buffering substances and the like. The pharmaceutical carrier employed will thus vary depending on the route of administration. Carriers may enable the pharmaceutical compositions to be formulated into tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions to aid intake by the patient. A thorough discussion of pharmaceutically acceptable carriers is available in Remington's Pharmaceutical Sciences (Mack Pub. Co., N.J. 1991).
The agent may be delivered by any known route of administration. The agent may be delivered by a parenteral route (e.g. by injection, either subcutaneously, intraperitoneally, intravenously or intramuscularly or delivered to the interstitial space of a tissue). The compositions can also be administered into a lesion. Other modes of administration include oral and pulmonary administration, suppositories, and transdermal or transcutaneous applications, needles, and hyposprays.
The agent that binds C5 may be administered alone or as part of a treatment regimen also involving the administration of other drugs currently used in the treatment of patients with myasthenia gravis. For example, the agent may be administered in combination with anticholinesterase agents, such as neostigmine and pyridostigmine, or immunosuppressive drugs, such as prednisone, cyclosporine, and azathioprine. Combinations of drug treatments may have an additive or synergistic effect on treatment of the disease.
The invention thus provides (i) an agent that binds C5, preferably the EV576 protein or a functional equivalent thereof, and (ii) an anticholinesterase agent and/or an immunosuppressive drug, for use in therapy.
The invention also provides the use of—(i) an agent that binds C5, preferably the EV576 protein or a functional equivalent thereof, and (ii) an anticholinesterase agent and/or an immunosuppressive drug, in the manufacture of a medicament for treating myasthenia gravis.
The agent that binds C5 may be administered simultaneously, sequentially or separately with the other drug(s). For example, the agent that binds C5 may be administered before or after administration of the other drug(s).
The invention thus provides the use of an agent that binds C5, preferably the EV576 protein or a functional equivalent thereof, in the manufacture of a medicament for treating myasthenia gravis in a subject, wherein said subject has been pre-treated with an anticholinesterase agent and/or an immunosuppressive drug. The invention also provides the use of an anticholinesterase agent and/or an immunosuppressive drug in the manufacture of a medicament for treating myasthenia gravis in a subject wherein said subject has been pre-treated with an agent that binds C5, preferably the EV576 protein or a functional equivalent thereof.
The agent that binds C5 may also be administered as part of a treatment regimen also involving the administration of other drugs currently used in the treatment of other diseases with which myasthenia gravis is associated, such as a thymic tumor, thyrotoxicosis, rheumatoid arthritis and lupus erythematosus. The agent that binds C5 may be administered simultaneously, sequentially or separately with the other drug(s). For example, the agent that binds C5 may be administered before or after administration of the other drug(s).
Various aspects and embodiments of the present invention will now be described in more detail by way of example. It will be appreciated that modification of detail may be made without departing from the scope of the invention.
1. Mechanism of Action and Inhibitory Concentration.
EV576 was purified from salivary gland extracts of the soft tick Orthinodoros moubata by SDS-PAGE and RP-HPLC of fractions of salivary gland extract found to contain complement inhibitory activity by classical haemolytic assays (
EV576 inhibits both human and guinea pig classical and alternative pathways. It has no effect on the rate of C3a production (
The ability of EV576 to inhibit both the classical and the alternative complement pathways is due to binding of the molecule to complement C5, the precursor of C5a and C5b-9. EV576 binds directly to C5 (
Recombinant EV576 (rEV576) with glycosylation sites removed (which otherwise are glycosylated in the yeast expression system) is as active as the native non-glycosylated protein (
The structure of EV576 confirms that it is an outlying member of the lipocalin family (
2. Half-Life.
The serum beta half-life of 125I labelled rEV576 in rats was found to be ≈30-38 hours.
3. Effect of EV576 on Experimental Autoimmune Myasthenia Gravis.
Experiment 1
Experimental autoimmune myasthenia gravis (EAMG) was induced in female Lewis rats according to the method of Piddlesden et al. (supra).
Lewis rats were injected with 1 mg/kg anti-AchR mAb35 intraperitoneally at day 0, along with either: i) 3.25 mg rEV576 (calculated to be 2.5× the molar dose needed to bind all available C5); or ii) phosphate buffered saline (PBS). The rats were assessed for changes in weight and clinical score over 7 days. (
Injection of 3.25 mg rEV576 totally attenuated mAb35 induced weight loss and muscular weakness compared to control.
All control animals became moribund and had to be euthanased at 72 hours post-induction whereas all rEV576 treated animals survived, gained weight and showed no muscle weakness for the duration of the experiment (183 hours) (
A single injection of rEV576 thus completely attenuates the symptoms of EAMG for at least 7 days.
Experiment 2
rEV576 was further analysed in a chronic model of EAMG. In this model, animals receive acetylcholine receptor protein in Complete Freund's Adjuvant (CFA) and generate their own antibodies over the course of approximately 30 days. This model mimics the human condition where symptoms occur and progress relatively slowly.
18 Lewis rats were immunised by subcutaneous injection of purified acetylecholine receptor protein (2×20 μg in 100 μl of PBS emulsified in equal volume of complete Freund's adjuvant+nonviable Mycobacterium tuberculosis−0.5 mg). Control rats were injected with PBS only.
After the onset of disease (EAMG clinical score 1 or 2 and weight loss<15%) 9 rats were injected with 3.25 mg of rEV576 i.p. and treatment continued for 10 days with 1 mg for every 12 hrs. Out of these nine rats, three rats were assessed as having severe myasthenia (S-EAMG) and six rats as having mild myasthenia (M-EAMG).
The remaining 9 rats were untreated. Out of these nine rats, four rats were assessed as having severe myasthenia (S-EAMG) and five rats as having mild myasthenia (M-EAMG).
The two sets of rats were each assessed for changes in weight, grip strength and clinical score over 10 days.
The rats in the severe EAMG group had lost 12% of their body weight and were within 24 hours of death when treatment with rEV576 was started. Untreated rats all died within 24 hours. The rEV576 treated rats showed a significant reduction in weight loss. At the beginning of treatment with rEV576 on Day 33, the average clinical score was 2.0. Injection of rEV576 for 10 days reduced the severity of clinical symptoms and prevented further weight loss (
In rats exhibiting the mild early stage of EAMG, clinical signs were just starting and weight was just starting to decrease at Day 33. Weight loss was prevented in rats treated with rEV576 and rats gained in average about 3.43% of body weight. Untreated animals with identical clinical scores lost about 4.59% of body weight (
Blood was collected from the rats for the detection of AchR antibodies and an assessment of complement hemolytic activity. There was no difference between experimental groups in the AchR antibodies detected by direct ELISA (
In addition, the cytotoxicity of rat serum from rats exhibiting both severe and mild EAMG was measured on rhabdomyosarcoma cell line (ATCC, CCL-136) using a ToxiLight Bioassay Kit. As can be seen in
This data suggests that rEV576 may be effective in treating both early mild myasthenia gravis and severe late stage disease (eg myasthenic crises).
| Number | Date | Country | Kind |
|---|---|---|---|
| 0518443.7 | Sep 2005 | GB | national |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/GB2006/003265 | 9/5/2006 | WO | 00 | 3/30/2009 |
| Publishing Document | Publishing Date | Country | Kind |
|---|---|---|---|
| WO2007/028968 | 3/15/2007 | WO | A |
| Number | Name | Date | Kind |
|---|---|---|---|
| 5041389 | Lindstrom | Aug 1991 | A |
| 5858969 | Marsh et al. | Jan 1999 | A |
| 6103748 | Bryan | Aug 2000 | A |
| 7884066 | Ting | Feb 2011 | B2 |
| 7884069 | Schaebitz et al. | Feb 2011 | B2 |
| 20040014782 | Krause | Jan 2004 | A1 |
| 20070048248 | Benedict et al. | Mar 2007 | A1 |
| 20070141573 | Nunn | Jun 2007 | A1 |
| 20080114055 | Kiss | May 2008 | A1 |
| 20080199484 | Yu et al. | Aug 2008 | A1 |
| 20100105611 | Hamer | Apr 2010 | A1 |
| 20110059885 | Lea et al. | Mar 2011 | A1 |
| Number | Date | Country |
|---|---|---|
| WO-9317099 | Sep 1993 | WO |
| WO2004106369 | Dec 2004 | WO |
| 2005023866 | Mar 2005 | WO |
| 2005079363 | Sep 2005 | WO |
| WO-2007028968 | Mar 2007 | WO |
| WO-2007117241 | Oct 2007 | WO |
| WO-2008029167 | Mar 2008 | WO |
| WO-2008029169 | Mar 2008 | WO |
| WO-2009098454 | Aug 2009 | WO |
| Entry |
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| Number | Date | Country | |
|---|---|---|---|
| 20090209459 A1 | Aug 2009 | US |
