WO2006014911A2 - Traitement de troubles inflammatoires, auto-immunes ou autres a l'aide d'agents qui permettent de reduire la sequestration du zinc par la calprotectine - Google Patents

Traitement de troubles inflammatoires, auto-immunes ou autres a l'aide d'agents qui permettent de reduire la sequestration du zinc par la calprotectine Download PDF

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WO2006014911A2
WO2006014911A2 PCT/US2005/026413 US2005026413W WO2006014911A2 WO 2006014911 A2 WO2006014911 A2 WO 2006014911A2 US 2005026413 W US2005026413 W US 2005026413W WO 2006014911 A2 WO2006014911 A2 WO 2006014911A2
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calprotectin
zinc
activity
localized
molecules
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PCT/US2005/026413
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WO2006014911A3 (fr
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David C. Kossor
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Kossor David C
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • A61K31/19—Carboxylic acids, e.g. valproic acid
    • A61K31/195—Carboxylic acids, e.g. valproic acid having an amino group

Definitions

  • the invention relates to biochemistry and medicine, and to a protein called calprotectin, which binds to calcium and zinc in body fluids. It has been used in the past as a marker and diagnostic indicator for certain diseases.
  • This invention discloses therapeutic interventions that can control calprotectin activity in various diseases, such as some autoimmune diseases.
  • Calprotectin is one of several names given to a certain protein that, under normal conditions, helps humans or other mammals fight bacterial infections. Because this protein became of interest to a number of research teams that approached it from different angles, and because it is made up of two polypeptide subunits that belong to a known family of polypeptides, calprotectin and its subunits have been given a number of different names, which require a brief listing and summary.
  • calprotectin is formed from two different subunits that bind to each other. That binding reaction is not covalent; instead, it is comparable to an antibody binding to an antigen. Since the two subunits are different from each other, calprotectin is considered and described as a "hetero-dimer" . There also is evidence that some calprotectin contains two copies of the light subunit and one copy of the heavy subunit, and still other calprotectin apparently contains two copies of each subunit.
  • Both of the two subunits and their genes were fully sequenced by the late 1980' s (Odink et al 1987; Lagasse et al 1988; Andersson et al 1988), and their crystalline structure has been determined (Itou et al 2001 and 2002; also see Moncrief et al 1990, Raftery et al 1996 and 1999, Loomans et al 1998, and Rety 2000 for additional information on the folding, conformation, and structure of the subunits). Both subunits belong to a class of polypeptides called SlOO peptides.
  • the lighter subunit which has 93 amino acid residues ll kilodaltons (kDa), usually is called S100A8, but it is also called the MRP8 protein (MRP is derived from the phrase "migration inhibitory factor related protein"), the LlL protein (derived from leukocyte-derived light chain), or calgranulin A.
  • MRP8 protein MRP is derived from the phrase "migration inhibitory factor related protein"
  • LlL protein derived from leukocyte-derived light chain
  • calgranulin A The heavier subunit, which has 114 amino acid residues and a molecular weight of about 14 kilodaltons (kDa) usually is called S100A9, but it is also called the MRP14 peptide, the LlH peptide, or calgranulin B.
  • the complete protein, formed when two or more subunits bind to each other, is called calprotectin, the S100A8/S100A9 protein, the MRP8/14 protein, or the 27E10 antigen. It is also sometimes called "the cystic fibrosis antigen"; however, the S100A8 (MRP8) and the S100A9 (MRP14) subunits also apparently have been referred to in various articles as the cystic fibrosis antigen.
  • Calprotectin plays an important role in a mammalian system that is usually called the innate immune system. That term can be understood by considering how and why it is different from the adaptive immune system.
  • the adaptive immune system requires and uses antibodies, which are made and used with the involvement of various white blood cell types, including B cells, T cells, macrophages, killer cells, etc.
  • Antibodies are proteins with variable sequences, created by complex genetic rearrangements within the chromosomes of certain types of white blood cells.
  • a wide assortment of antibodies is effectively thrown up against a population of invading bacteria, viruses, or other microbes, and some of those antibodies will bind to proteins on the surfaces of the microbes.
  • Those antibodies that bind to the invading microbes will be identified, selected, and then mass-produced, by means of still more complex processes.
  • the process of generating an assortment of antibodies, selecting specific antibodies that have managed to bind to a specific type of invading microbe, and mass-producing the antibodies that happen to be effective in combatting a particular infection usually takes several days. That delay can allow most types of bacteria and viruses (which can reproduce many times faster than mammalian cells) to generate huge numbers of invading microbes, before a complete defensive response that requires antibodies can move rally into action. That delay, before the adaptive immune system can mount an effective full-scale defense, is why vaccines (if prepared and administered in ways that allow the immune system to get ready, in advance, for an infection), can make a huge difference in how severe a disease or infection will become.
  • the defensive molecules that can respond immediately to invading microbes, and the cells that carry them around and release them at sites of infection are called the "innate" immune system.
  • This system is also regarded by some as a “primitive” immune system, and it is still the main line of defense among some types of insects, worms, and other lower animals that do not have immune systems with antibodies.
  • Calprotectin The innate immune system in humans and other mammals includes calprotectin, which is carried in very high concentrations by white blood cells called “neutrophils". Calprotectin makes up an estimated 45% to 60% of all the water-soluble proteins carried by neutrophils. Its antimicrobial activities are discussed in articles such as Steinbakk et al 1990, and Sohnle et al 1991, 2000a, and 2000b.
  • neutrophil cells When neutrophil cells are attracted to the site of an infection, they release their calprotectin. The calprotectin then grabs and "sequesters" any available zinc (i.e. , it binds tightly to any available zinc ions, in a way that renders the zinc unavailable to the invading microbes) .
  • the calprotectin grabs and "sequesters" any available zinc (i.e. , it binds tightly to any available zinc ions, in a way that renders the zinc unavailable to the invading microbes) .
  • An understanding of the importance of this process requires some background information on the roles and actions of zinc, in mammalian physiology.
  • zinc exists in either "free” form (which usually is in the form of positively charged ions, Zn + + ), or in "bound” forms in which it is associated with proteins or other molecules.
  • "Free” zinc can also be referred to by terms such as unbound, exchangeable, rapidly exchangeable, available, accessible, chelatable, bindable, labile, etc. It also needs to be recognized that even in "bound” forms of zinc, the strength of the binding can range from relatively loose, to very tight. As a result, free and bound zinc will generally establish a dynamic and adaptive equilibrium that can change in various fluid or cell types, and over time. P L.
  • zinc also helps create the catalytic activity that is essential for many enzymes. In general, it does this by participating in various types of electron transfers, which are essential for breaking old bonds and forming new bonds.
  • available zinc is enormously important to all cells, including both (i) invading microbes, and (ii) the cells of an animal host. Therefore, when calprotectin that has been released by neutrophil cells of a mammal, as a rapid defensive mechanism, binds tightly to and sequesters any available zinc in a localized area or segment of tissue, the available zinc cannot be used by invading microbes. This prevents the microbes from being able to obtain enough zinc to grow and reproduce at uncontrolled rates.
  • This "first response" defense helps keep an infection under control, while a larger antibody response is being prepared. This is described in more detail in articles such as Striz et al 2004, a review article with numerous other articles cited therein.
  • calprotectin Although the "innate immune" functions of calprotectin are essential for helping mammals defend against invading microbes, researchers have recognized in recent years that some types and cases of inflammatory conditions (such as rheumatoid arthritis, multiple sclerosis, inflammatory dermatoses, cystic fibrosis, and certain other disorders) are characterized by excessive amounts of calprotectin. Consequently, high levels of calprotectin in certain types of disorders are being used and/or evaluated as a marker, fi1N5 ⁇ cltof, i '-4 ⁇ l ⁇ oSt ⁇ fl
  • calprotectin Although increased concentrations of calprotectin are being used as diagnostic agents (or markers, biomarkers, etc.) to assess the severity of various inflammatory diseases, cancers, etc., the consequences of the increased local sequestration of zinc by calprotectin has not been evaluated, and it is believed by the Inventor herein that in some cases, in some patients, conditions are being created where calprotectin responses and activities reach excessive levels that begin to starve local areas or types of tissues of zinc.
  • Agren 1990 reported that in lab animals, topically-applied zinc accelerated the growth of epidermal cells and the closure and healing of experimentally-inflicted cuts, and Other reports which indicate that zinc can help accelerate the healing of skin wounds, skin ulcers in diabetics, etc. , include Pories et al 1967, Husain 1969, and Hallbook et al 1972, Stromberg et al 1984, and Apelqvist et al 1990.
  • zinc has ⁇ p ⁇ Ma%ll ⁇ H ⁇ ve Ingi ⁇ teiiti ⁇ n. ointments for treating diaper rash, for decades.
  • metalloproteinases which play crucial roles in manipulating and "remodeling" collagen, the fibrous protein that holds together cells in connective tissues, in animals.
  • Collagen fibers that are more than a few weeks old are constantly being degraded, so they can be replaced by new collagen, which is gradually secreted by various types of cells. This is a major process in all higher animals; in humans, it enables muscles and connective tissue to remain strong and flexible over a span of numerous decades.
  • the degradation and removal of old collagen, and its replacement by new collagen require direct and continuing involvement by metalloproteinases, and those enzymes simply cannot function without zinc.
  • metalloproteinase defects or alterations are directly involved in a number of diseases, including angiogenesis and the growth of cancerous tumors, lung diseases, and myocardial problems.
  • metalloproteinase enzymes in connective tissues, and in various diseases, are described in articles such as Woessner 1991, Isakson et al 2001, and Pardo et al 2005. Since zinc is essential for metalloproteinase enzyme activity, localized zinc deficiencies can pose risks of disrupting the maintenance and gradual turnover of collagen fibers, in connective tissues.
  • homeostasis is a medical term that refers to a dynamic and adaptive equilibrium; an animal body attempts to maintain its homeostasis, despite fluctuations in food intake, outside factors, etc.).
  • the tissues involved will be able to tolerate any temporary and transitory localized decreases in zinc that may be caused by calprotectin release, and can reestablish healthy and appropriate levels of zinc in ways that will avoid and minimize any tissue injury.
  • references herein to terms such as therapy, treatments, etc. are limited to interventions that reduce calprotectin levels, and that help relieve suffering or discomfort and improve the health, quality of life, or similar conditions of a patient in need of such treatment, by reducing the severity, retarding the progression, or alleviating the symptoms of a disease or disorder. It has been known for years that calprotectin concentrations in feces or body fluids can be used in diagnostic methods for measuring and monitoring various diseases. However, this current invention does not relate to diagnosis, measuring, or monitoring of diseases; instead, it is limited to therapy and treatment of diseases.
  • disease includes conditions that are often referred to as disorders, syndromes, or similar terms by medical professionals. This excludes traumas and other external injuries, and it excludes microbial infections. However, diseases can and often do arise from bodily repair processes that have gone awry in ways that create or aggravate a chronic, degenerative, or similar problem when the body attempts to recover from a trauma or infection.
  • disorder refers to an underlying biochemical problem that causes or aggravates a disease as defined above.
  • these disorders involve (usually as a triggering or aggravating factor, or as a link in a chain or cascade) a Pa'!f)fSpCtta ⁇ &trM[0!ffiflf ⁇ Usichronically elevated, in a manner that leads to localized deficits in available zinc concentrations.
  • the term "patient” refers to a person who is suffering from a calprotectin-related disease or disorder as described herein, or who can substantially benefit from a treatment as disclosed herein, regardless of whether such person actually seeks medical attention or treatment.
  • Medical attention is not limited to treatment by licensed physicians; instead, it refers to a treatment that can address a problem that can and should properly be regarded as a medical problem.
  • people who suffer from various minor aches and pains and friends, family, neighbors, pharmacists, nurses and other caregivers, etc.
  • people who suffer from various minor aches and pains often can recognize the symptoms of a problem, and can use or recommend helpful yet relatively low-cost treatments (such as, for example, over-the-counter treatments that can be purchased in drugstores) without requiring a visit to a physician.
  • references to "local” (or localized, etc.) zinc deficiencies are not intended in a strict manner, and instead can include nearly any type or level of zinc deficiency that is not system- wide.
  • cystic fibrosis directly affects the lungs; it is less well known that it also affects the liver. Accordingly, calprotectin over- expression and zinc deficiencies in cystic fibrosis patients may be affecting both the liver and the lungs, in various patients. Such effects are regarded as localized, as that term is used herein.
  • rheumatoid arthritis tends to attack articulating joints with cartilage segments. Accordingly, a problem that arises in multiple body parts that share certain traits (such as articulating joints, which contain cartilage segments), would be regarded herein as a problem in local tissues, rather than a systemic problem, even if the "common trait" tissues occur in various distributed tissues located in different parts of the body.
  • Calprotectin molecules that have zinc binding sites that are wn-occupied, and that are therefore ready to receive and chelate zinc ions are referred to as active calprotectin.
  • active calprotectin calprotectin molecules with zinc binding sites that are already occupied (or saturated, etc.), either by zinc or by "calprotectin suppressing drugs" (CSD's), as described below, are referred to as inactive (or inactivated) calprotectin, since they no longer have the ability to bind to zinc ions.
  • CSD's calprotectin suppressing drugs
  • one object of this invention is to disclose methods and agents for tihSapeu ' tkf SeSrSenf
  • Another object of this invention is to disclose that "targeted" methods for reducing concentrations or activity levels of calprotectin in specific localized tissues can provide effective ways for treating various disorders inflammatory or other disorders that are caused, aggravated, or otherwise related to or characterized by elevated concentrations of calprotectin.
  • Another object of this invention is to disclose that once the "targeted" methods for reducing concentrations or activity levels of calprotectin in specific localized tissues have been shown to be effective, in "proof of principle” tests, screening methods can be used to identify nonprotein small-molecule "calprotectin-suppressing drugs" (CSD 's) that will either: (i) bind to calprotectin in ways that will block, reduce, or otherwise modulate the sequestering of zinc by excess calprotectin, or (ii) help suppress the release of calprotectin molecules, by neutrophil cells. Either of those two approaches can provide therapeutic benefits for at least some inflammatory or other disorders that are aggravated by elevated concentrations of calprotectin.
  • CSD nonprotein small-molecule "calprotectin-suppressing drugs”
  • calprotectin a protein normally carried by leukocytes.
  • calprotectin is released by leukocytes at the site of a microbial infection. It provide a first line of defense against microbial growth, by sequestering any available zinc. Since zinc is essential for microbial growth and reproduction, this initial response helps control and limit an infection, while a complete antibody response is being generated.
  • derangements of the calprotectin defense mechanism hold an important key to at least some types and/or cases of autoimmune and/ or inflammatory disorders, arising from local or regional deficiencies of zinc.
  • zinc is essential to cell reproduction, tissue growth, and numerous enzyme activities, and it plays P 1I liiiiar ' iyi& fi:lthe-V ⁇ i ⁇ !- 4i i ⁇ & . ⁇ fealing of damaged tissues. Therefore, local or regional deficiencies of zinc, caused by excessive activity of calprotectin, can seriously hinder the natural repair mechanisms that healthy tissues use to keep inflammatory outbreaks under proper regulation and control.
  • two different approaches are disclosed herein, which can use currently available devices, methods, and reagents to provide targeted forms of therapy that will aim and focus their effects on specific targeted organs, limbs, or tissue types that are indeed suffering from localized zinc deficiencies, which are being caused by chronic calprotectin surpluses.
  • One approach involves arterial infusion of a liquid that contains a zinc buffer, directly into an artery that supplies affected tissues.
  • the liquid will provide sufficient quantities of zinc (in a suitable transport or buffer compound) to saturate any active calprotectin molecules in the region, thereby converting them into zinc-saturated and therefore inactive calprotectin molecules.
  • the liquid will then provide additional zinc to the with zinc deficits caused by excess levels of active calprotectin.
  • extra-corporeal blood processing also called “plasmapheresis”
  • plasma or serum is passed through a device (such as an affinity column containing monoclonal antibodies) that removes calprotectin from the plasma or serum.
  • a device such as an affinity column containing monoclonal antibodies
  • Either or both of these approaches can provide therapeutic benefits, using currently available technology; and, for some patients with certain disorders (such as inflammatory conditions that may completely resolve if the affected tissues are given a chance to heal), reasonably short periods of treatment may be able to provide lasting benefits and possibly even permanent cures.
  • Cystic fibrosis F'-l ⁇ T/s ⁇ Siffiiiat ⁇ ii/.Edife ⁇ iktBses that are characterized by surplus calprotectin levels, including psoriasis and some types of atopic dermatitis;
  • NO's nitrous or nitric oxides
  • Cancers that are characterized by elevated calprotectin levels which includes some cases of colon or other gastrointestinal cancers, some liver and hepatobiliary cancers, some carcinomas of the head or neck, some cancers of the oral cavity, breast, pancreas, lungs, or ovaries, and some types of lymphomas and leukemias;
  • Liver diseases that are characterized by surplus calprotectin levels, including primary biliary cirrhosis (an autoimmune liver disorder that damages the septal and intrahepatic bile ducts, leading to fibrosis and other problems) and primary sclerosing cholangitis; and,
  • NO's Various other diseases that involve apparent or inducible increases in nitrous or nitric oxides (collectively referred to as NO's, or occasionally as NOx or NOX where X is a variable that indicates that the compound(s) of interest might include any or all of NO, NO 2 , NO 3 , and possibly even N 2 O) in localized tissues, or that are characterized by elevated levels of an enzyme called inducible nitric oxide synthase (iNOS); in particular, disorders that involve NO are of interest, because NO has an unstable and reactive "resonant" structure that is known to create or aggravate various inflammatory conditions.
  • iNOS inducible nitric oxide synthase
  • the treatments disclosed herein merit evaluation for their potential ability to help prevent or reduce various neurological diseases, potentially including Alzheimer's disease, dementia, multiple sclerosis, and autism. It is worth noting, in particular, that some case reports have suggested that the apparent onset of autism, in some small children, was preceded by a severe bout of gastrointestinal distress. This raises questions as to whether such episodes may have triggered transitory calprotectin over- expression levels, and transitory zinc deficits, that may have inflicted lasting and even permanent damage on neonates or infants because they occurred during critical growth stages.
  • agents that can competitively bind to calprotectin can help reduce the tendency of excessively high calprotectin levels, in inflamed or otherwise stressed tissues, to create or aggravate local or regional zinc deficiencies that cause or aggravate inflammatory, autoimmune, or other disease processes.
  • Targeted Injections or Infusions F C T/rM 'SrSt ffiethcBE ⁇ fcL ⁇ r ⁇ fes targeted injections of a blood-compatible aqueous liquid (such as Ringer's lactate, a solution of glucose in buffered saline, etc.) that contains high concentrations of zinc, carried by a suitable transport or "buffer" compound, directly into an artery that supplies blood to localized tissues that are suffering from a calprotectin surplus and/or zinc deficit,
  • a blood-compatible aqueous liquid such as Ringer's lactate, a solution of glucose in buffered saline, etc.
  • blood from one or more veins that carry blood out of the targeted localized tissue also can be chemically processed, to remove any unused or surplus zinc from the blood (or diluted blood) that emerges from the tissue being treated.
  • the surplus free zinc in the injected blood mixture or other liquid will bind to the zinc-binding sites of the surplus calprotectin in the affected tissue, to a point that will saturate the zinc-binding sites of the calprotectin. This will prevent the calprotectin molecules from chelating and effectively withdrawing additional free zinc from the blood, lymph, or other liquids that surround and bathe the affected tissues. In addition, any remaining zinc that was supplied by the injected liquid can directly benefit the tissue that previously had been deprived of sufficient zinc, due to calprotectin' s chelating activities.
  • a preferred method can be performed by using a slower infusion, over a span of, for example, one to several hours, in a manner comparable to a dialysis treatment. It also may be possible to develop and use "shunt" devices, analogous to shunts used in dialysis, to enable repeated infusions of a zinc-supplemented liquid into a particular targeted artery, over a span of multiple weeks or months. This type of approach also likely can be adapted to at-home and/or mobile treatments, which have become available for dialysis patients through the development of dialysis machines that are roughly the size of large briefcases, and that can be operated at home.
  • targeted zinc infusions may be able to help PdSfl&g €diisst ⁇ feg'aiil ⁇ ihlei ⁇ hiproved "homeostatic set-point", which may effectively comprise a state of health, mid-term or long-term remission, etc. In some cases, this may be able to completely eliminate the need for subsequent infusions, after localized damaged tissue has been effectively repaired. In other cases, a relatively intense round of frequent, high-dosage, or other "restorative" treatments may be able do sufficient good to allow any subsequent "maintenance" infusions to be reduced to a relatively infrequent basis, such as once a week, once a month, etc.
  • Candidate compounds that offer good candidates for evaluation as transport or buffer compounds, as described above, include complexes formed by reacting zinc with relatively weak organic acids, to form zinc gluconate, citrate, picolinate, etc.
  • Another method of treatment that can be carried out using already-known and available methods, machines, and reagents comprises a process that is usually called plasmapheresis, or extra-corporeal blood processing. Briefly, it involves the following steps:
  • the processing device can be an affinity column that has been loaded with a monoclonal antibody preparation.
  • the monoclonal antibodies will bind to calprotectin, and they will be chemically affixed to beads that are trapped inside the column by filter screens at the inlet and outlet of the column;
  • plasmapheresis i.e., extra-corporeal blood processing
  • plasmapheresis can remove calprotectin-loaded blood from a vein, pass it through a monoclonal antibody column or other device that will remove calprotectin from the blood, and then return the calprotectin- free blood to the patient's body.
  • extra-corporeal blood processing can be used to reduce the numbers of neutrophil cells that are being sent to a localized tissue area that is suffering from a calprotectin surplus.
  • This type of processing can use, for example, monoclonal antibodies that bind to certain antigens that are known to exist in abnormally large numbers on the surfaces of neutrophil cells.
  • antigens are well-known, and are referred to as "human neutrophil antigens" (HNA's), as reviewed in Stroncek 2004.
  • platelets also called megakaryocytes
  • platelets are specialized types of blood cells, and are heavily involved in blood clotting.
  • platelets are isolated from blood provided by blood donors, and are made available for transfusions into hemophiliacs and others.
  • the methods that are used to isolate platelets may be adaptable for removing neutrophils from the blood of people who are suffering from diseases that involve excessive calprotectin activity and local zinc deficits.
  • Either or both of the two methods summarized above can be used: (i) to directly confirm that calprotectin-reducing treatments can substantially benefit patients who are suffering from various diseases, such as the diseases listed above; and, (ii) to identify particular diseases and patient subpopulations that demonstrate the greatest benefits and improvements from such treatments.
  • the ultimate goal and objective of screening programs for identifying compounds that have CSD activity will be to identify not just one, but ultimately two different classes of nonprotein CSD drugs.
  • One class of such drugs will bind strongly and competitively to the zinc binding sites of calprotectin molecules that have been released by neutrophil cells, thereby occupying those zinc binding sites; this will inactivate those calprotectin molecules, by rendering them unable to sequester any additional zinc that may be available.
  • the other class of desirable drugs will suppress the release of calprotectin molecules, by neutrophil cells that carry calprotectin. Either of those two classes of drugs can be highly useful, on its own, in helping reduce and control the localized zinc deficits that arise from excessive calprotectin levels in various types of diseases.
  • both drugs acting simultaneously, may be able to offer additive or synergistic benefits that, when combined, may be substantially more effective than either drug can achieve by itself. Accordingly, identification of drug candidates that can achieve either of those two different activities (suppression of calprotectin release by neutrophil cells, or suppression of zinc binding by already-released calprotectin molecules) should be regarded as useful and valuable goals and objectives of such screening programs.
  • this invention discloses various factors and insights that can help guide the screening, identification, development, and Qel&ngfJfrn6tpoteafi ⁇ ii
  • calprotectin-binding polypeptide molecules are highly useful as research reagents, and they are specifically claimed herein, as compositions of matter, because of their utility in that field. They can be manufactured and sold, as an item of commerce, to laboratories, in a manner comparable to other research reagents. Furthermore, under some circumstances, they may turn out to be useful and valuable as human therapeutic agents, in a manner comparable to injectable insulin preparations.
  • polypeptides are less preferred for medical use, than non-protein drugs (often referred to as "small molecule” drugs).
  • the screening criterion is simple, straightforward, and clear: the goal is to find and identify agents that can bind competitively to the zinc-binding sites of the calprotectin protein.
  • calprotectin is a protein that has been known for decades, formed from two subunits that have been fully sequenced (as reported in Odink et al 1987 and Lagasse et al 1988), an automated screening program using computerized machinery that requires very little manpower or active supervision can be set up and run, using procedures that are well-known to those who specialize in designing and running such screening efforts.
  • this type of meter can rapidly measure free zinc levels, and express them in a logarithmic "pZn" scale, comparable to the standard pH scale for acidity.
  • This meter will enable physicians and researchers (or even patients, when offered for at-home use, in a manner comparable to glucose monitors for diabetics) to detect and monitor changes in available zinc in liquid or other samples (such as blood, urine, tissue biopsy samples, etc.) obtained from sites that manifest or are correlated with an inflammatory or other relevant disease process.
  • concentrations of free zinc in various liquids can be rapidly measured, and utilized as an effective marker or indicator, both for screening programs at pharmaceutical companies, as described above, and for evaluating the efficacy of treatment regimens that are being tested or used, in animal test, human clinical trials, or medical or veterinary use.
  • free zinc meters can allow physicians, researchers, and patients to rapidly and conveniently determine: (i) the most appropriate therapeutic agent to administer, when treatment commences; (ii) the optimal dosages of a selected agent to be administered at any jSMicula/ Mfe ⁇ GSf ⁇ i. ⁇ ' v ⁇ ifcilitHb ⁇ tfentially changing severity of a particular case of a particular disease, and (ii) appropriate adjustments (in dosages, chosen agents, etc.) to maintain optimal effectiveness of a therapeutic regimen, in a specific patient, over a span of months or years.
  • a typical cell-free competitive binding assay can be carried out by steps such as the following:
  • a purified preparation of calprotectin molecules is created. This can be done by using either of two main approaches, and both approaches should be tested and compared against each other, to confirm their validity.
  • One method involves obtaining a supply of calprotectin from human neutrophils or other cell types (including, for example, certain types of reported transformed cell lines that can reproduce indefinitely, in cell culture conditions, and that can be induced to secrete or release calprotectin).
  • the other main approach involves the fermentation of E. coli, yeast, or other host cells that have been transfected by plasmids or other vectors that carry genes that will express the two calprotectin subunit polypeptides, to obtain "recombinant" calprotectin that has the same amino acid sequence as the version formed in human neutrophils.
  • Either type of supply can use any of various known separation methods (such as, for example, electrophoresis, isoelectric focusing, or an affinity column containing monoclonal antibodies that bind to calprotectin) to purify the calprotect
  • the beads are washed or rinsed, to remove any non-immobilized calprotectin molecules from the bead preparation, and the bead preparation is leaded into a suitable type of column with filters at the inlet and outlet ends.
  • a "binding buffer” is passed through the column, to equilibrate the column and establish conditions of temperature, acidity, and salinity that will promote the binding of various candidate drug to the immobilized calprotectin molecules;
  • a confirmatory and calibrating binding step is carried out, using a mixture that contains a reagent such as radiolabeled zinc isotopes, to ensure that the reaction that was used to affix the calprotectin molecules to the beads did not alter the zinc binding sites or render them inaccessible.
  • a reagent such as radiolabeled zinc isotopes
  • This "calibration” step can also be used to determine various additional factors, such as (i) the total binding capacity of the column, under various conditions of temperature, acidity, and/or salinity (which can then be altered or controlled, if desired, to modify that binding capacity); and, (ii) the percentage of a fixed quantity of a certain zinc isotope that will bind inside the column, when no competing drug candidates have been passed through the column.
  • isotopes of zinc are known, and each has its own type of emission, which can be measured by a corresponding type of detector.
  • the 65 Zn isotope emits gamma rays.
  • Gamma rays pose a danger to personnel, and 65 Zn has a half-life of about 273 days; these two factors, taken together, make 65 Zn very expensive to dispose of, as a radioactive waste. Therefore, a positron-emitting isotope, 63 Zn, which has a half-life of only about 38 minutes, is safer and preferable for most types of research as disclosed herein.
  • P candidate calprotectin-binding drug carried by a binding buffer liquid, is passed through the column containing the immobilized calprotectin molecules. If the drug candidate has substantial affinity for the zinc-binding sites of the calprotectin, then the drug candidate will become and will remain affixed to the calprotectin, for as long as suitable binding conditions are sustained in the column;
  • a different buffer preparation containing free zinc (which can be labelled, if desired) is then passed through the column. If the zinc binding sites have been occupied by the candidate drug, smaller quantities of zinc will become bound to any remaining accessible zinc binding sites in the calprotectin molecules in the column, and larger quantities of zinc will simply pass through the column. Either or both of those zinc quantities can be measured. For example, if radiolabeled zinc is used, then the quantity of zinc that has become "stuck" inside the column can be measured; alternately, if mixtures containing unlabelled zinc are used, then the quantity of zinc that has emerged from the column, in the effluent, can be measured, by using the "free zinc meter" described above.
  • the quantity of zinc that passes through a column, after the column has received and processed a candidate drug preparation, is compared to the quantity of zinc that passed through a column when no candidate drug preparation had been passed through the column. If there is a large differential (i.e., if a substantially larger quantity of zinc passed rapidly through the column, after the column processed a candidate drug preparation) , then that differential indicates that a large number of zinc binding sites, on calprotectin molecules that remain immobilized within the column, were indeed occupied by the candidate drug; this means the candidate shows good potential, and deserves closer attention and more extensive testing. By contrast, if only a small differential is observed, it will indicate that only small numbers of zinc binding sites on the calprotectin molecules were occupied by the candidate drug, and that candidate drug does not have the desired competitive binding activity that is being sought.
  • the steps described above relate to one exemplary type of binding reaction that can be used for screening purposes.
  • Other types of competitive binding assays are also known, and can be evaluated for use as disclosed herein, if desired.
  • a system widely known as the BIACORETM system avoids the need for radioactive isotopes or other expensive chemicals, by using a method called "surface plasmon resonance spectroscopy", which involves alterations that occur when certain types of light are shown onto, and reflected from, thin surface films.
  • surface plasmon resonance spectroscopy which involves alterations that occur when certain types of light are shown onto, and reflected from, thin surface films.
  • IMAC Immobilized Metal Affinity Chromatography
  • this system would use zinc ions that are affixed (such as through a spacer chain) to a solid surface. Calprotectin molecules are preincubated with a candidate drug that may be able to bind the zinc-binding sites of the calprotectin. The calprotectin-drug mixture is then passed across the IMAC material. If the candidate drug became bound to the zinc-binding sites of the calprotectin, then the calprotectin molecules with their already-occupied binding sites will simply pass over the IMAC material, and emerge from the column or other device.
  • IMAC Immobilized Metal Affinity Chromatography
  • the calprotectin molecules will bind to the IMAC material. This allows a direct and convenient measurement of whether, and to what extent, a candidate drag will bind to the zinc-binding sites of calprotectin.
  • Candida albicans e.g., Sohnle et al 1991, 2000a, and 2000b.
  • a culture of C. albicans cells in a culture media that contains no zinc is inoculated with a second culture medium that contains zinc, but that also contains calprotectin, which has been preincubated with a candidate drag that is being screened.
  • the calprotectin will not sequester the zinc in the supplemental media, and the zinc will be available to the C. albicans cells, which will be able to grow.
  • the candidate drug did not bind to the zinc-binding sites of the calprotectin, then the calprotectin will sequester the zinc in the supplemental media, the zinc will not be available to the C. albicans cells, and the cells will not be able to grow.
  • albicans cells are highly susceptible to zinc levels, not just on a qualitative level (i.e., where the cells either grow or don't grow), but on a quantitative level, where the extent of cell growth (which can be measured by means such as glucose uptake levels, turbidity, colony sizes, etc.) is directly proportional to zinc concentrations, and provides a reliable and reproducible numerical indicator of how much available zinc remained in the supplemental culture media, after any "unoccupied" calprotectin molecules took away some portion of the zinc in the known media.
  • ACE inhibitors angiotensin converting enzyme
  • CAPOTEN a monopeptide converting enzyme
  • VASOTEC a monopeptide converting enzyme
  • MMP matrix metalloproteinases
  • any or all of the foregoing types of screening methods can be enhanced, if one or more known types of "starting point' or “baseline” compounds are known, and can be used as benchmarks, reference points, etc. This is indeed possible, because several specific compounds are already known to bind, with at least some level of affinity and specificity, to the zinc binding sites of calprotectin.
  • an interesting set of compounds that can offer potentially useful "baseline” compounds, in an effort to identify and develop better analogs comprises certain Bypesf ⁇ f
  • these compounds appear to inhibit zinc binding by calprotectin, to some extent (it should be noted that zinc binding was not measured directly, in the reports cited above; instead, those researchers measured apoptosis, which is dependent to some extent and in various ways on zinc concentrations).
  • TNF tumor necrosis factor
  • arachidonic acid an omega-6 fatty acid (Kerkhoff et al 1999a and 1999b, Klempt et al 1997), certain types of omega-3 fatty acids (Belluzzi et al 2000), and analogs of short polypeptide segments that have been isolated and sequenced from monoclonal antibodies that bind tightly to the zinc-binding sites of calprotectin.
  • starting material for preparing a library of analogs, variants, and other offshoots that branch out from that starting point, for a subsequent round of screening tests.
  • an essential starting material comprises a preparation of neutrophil cells that contain at least reasonably high levels of calprotectin molecules in their cytoplasm (i.e., the watery fluid that fills the cell).
  • Neutrophils that have been separated from blood from animals can be used, if desired; however, this can raise questions about whether the mechanisms and suppression of calprotectin by the neutrophils closely mimics and models the same processes involving human neutrophils.
  • ⁇ &T6oUttSiteiy ⁇ l ⁇ llSfu ⁇ lii ⁇ cancerous lines that are well known and widely available in research labs
  • neutrophil-producing lines by exposing them to certain types of hormones, such as "granulocyte-macrophage colony stimulating factor” (GM-CSF), which is sold by Amgen.
  • GM-CSF granulocyte-macrophage colony stimulating factor
  • blood marrow cells that create neutrophils can be converted (transformed) into immortal (cancerous) lines by various known methods, such as by contacting them with certain types of viruses.
  • Still more options involve creating "merged" cell lines, which will be comparable to the immortalized "hybridoma” cell lines that secrete monoclonal antibodies.
  • Hybridoma cell lines are created by using membrane-softening agents to merge cancerous lymphoma cells (which can reproduce endlessly) with B-cell lymphocytes that generate and secrete antibodies; after these cells have been merged together, progeny cells that happened to inherit immortalizing genes from the lymphoma cells, and antibody-producing genes from the B-cells) are identified, selected, and cloned.
  • immortalized cell lines that act like neutrophils in releasing calprotectin likely can also be created, if desired, by using similar methods.
  • Calprotectin release by such neutrophils in cell culture conditions, can be induced by contacting neutrophil cells that contain calprotectin with any of several known agents, such as a phorbol ester known as phorbol 12-myristate 13-acetate (PMA), as described in articles such as Kerkhoff et al 1999.
  • PMA phorbol 12-myristate 13-acetate
  • the phorbol ester will activate protein kinase C, a signalling protein that will then trigger a cascade that leads to the release of calprotectin.
  • these various reagents and cell types set the stage for screening assays that can be used to test candidate drug compounds, to determine whether (and how strongly) any particular candidate drug can suppress the release of calprotectin, by neutrophil cells.
  • One such set of steps that can be used to carry out such assays can be summarized as follows:
  • a candidate drug compound is preincubated with a population of neutrophils, at suitable conditions and for a suitable period of time.
  • the cells are then contacted by PMA or some other suitable triggering agent that stimulates calprotectin release.
  • the cell culture liquid is then tested, to determine how much calprotectin protein was released into the liquid, by the neutrophil cells.
  • This testing can be done in any of several ways.
  • Calprotectin protein concentrations can be measured directly, using any of ⁇ P €af ⁇ uslli ⁇ vll ! ⁇ nethdyS;' i yucih.-is an enzyme-linked immuno-sorbent assay (commonly known as an ELISA assay).
  • ELISA kits that have been developed specifically for measuring calprotectin are already commercially available, from CalPro-AS (Oslo, Norway; www.phical.com).
  • calprotectin levels can be measured indirectly, by measuring the levels of free zinc in a liquid, using the pZn meter that will soon become available from Neurobiotex, as described above (free zinc levels can indicate calprotectin levels, since any calprotectin released by the neutrophils will sequester and reduce the levels of free zinc in a liquid being measured).
  • any animal-derived or immortalized neutrophil (or neutrophil-like) cell line for such assays can be facilitated by testing the candidate cell types with any of several known drugs.
  • two drugs called amlexanox and cromolyn are known to permeate into neutrophil cells and bind to calprotectin molecules contained in the neutrophils, in a manner that impedes and suppresses the release of calprotectin by the neutrophils.
  • a method for treating a disease characterized by excessive levels of calprotectin activity in localized tissue comprising the step of reducing concentrations of active calprotectin molecules in said localized tissue, in a targeted manner that does not cause substantial alterations in zinc concentrations in a patient's stomach or intestines.
  • a method for treating a disease characterized by zinc deficits in ⁇ l(fc ⁇ lfzedltl ⁇ sUfe
  • the "targeted” administration can be accomplished by various means, such as (1) injection of said medicament into a body part that will cause transport of said medicament to said localized tissue, or (2) administering a medicament that has a specific binding affinity for calprotectin.
  • Nonprotein drugs and medicaments containing such drugs are also taught, for suppressing calprotectin activity and reducing zinc deficiencies in local tissue areas, wherein such nonprotein drugs have been identified by screening tests carried out on a molecular library, and the screening tests are designed to identify compounds that suppress calprotectin activity by either (1) binding to and occupying at least one zinc binding site in human calprotectin, or (2) suppressing calprotectin release by human neutrophil cells.
  • polypeptides that suppress calprotectin activity are taught, wherein the polypeptides are identified by screening of a molecular library to identify polypeptides that suppress calprotectin activity, either (1) by binding to and occupying at least one zinc binding site in human calprotectin, or (2) by suppressing calprotectin release by human neutrophil cells.
  • nutrikines may be able to help deliver zinc, in therapeutic concentrations, to localized or regional tissue locations that have been stressed by calprotectin-induced zinc deficiencies.
  • the term “nutrikines” refers to compounds that facilitate the specific delivery of a trace nutrient to a particular site (or a particular type of tissue) within the body. Nutrikines may be endogenous (e.g. originating in or produced by the body), or exogenous (e.g. originating or produced outside of the body).
  • Compounds that are believed to have certain properties that render them apparently capable of serving (to at least some extent) as zinc-transporting nutrikines are believed to include, for example, Protein Kinase C, melatonin (Dong et al 2003, Mei et al 2002), secretin, uroguanylin, cysteine-rich intestinal peptide (CRIP), the salivary histatin proteins, a salivary protein called gustin (also called carbonic anhydrase VI), and possibly certain types of carotenoids.
  • An example of a potential exogenous nutrikine is provided by the delivery of zinc to the liver (and in particular, to cells in and around the bile duct, in the liver, for treating various types of cancerous, hyperproliferative, or other conditions that affect the bile duct or surrounding tissues (e.g. , Dietz et al 1991).

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Abstract

L'invention concerne des traitements de types variés de troubles inflammatoires, auto-immunes ou autres caractérisés par un excès de calprotectine, protéine qui normalement protège contre les infections microbiennes par séquestration du zinc disponible au niveau d'un site d'infection. L'excès de calprotectine conduisant à des déficiences en zinc dans des tissus localisés peut créer ou aggraver certains troubles auto-immunes et/ou inflammatoires, mais les efforts pour fournir des compléments systémiques (oraux) de zinc activant des mécanismes de compensation sont inefficaces. De ce fait, l'invention concerne des traitements ciblés permettant de supprimer ou de contrôler l'excès de calprotectine dans des tissus locaux. Ces traitements comprennent des injections ciblées de solutions de zinc et un traitement par plasmaphérèse. L'invention concerne également des essais de criblage destinés à identifier des médicaments non protéiques qui peuvent i) soit se lier spécifiquement à des sites de calprotectine de liaison de zinc, ou ii) soit inhiber la libération de la calprotectine à l'aide de cellules neutrophiles.
PCT/US2005/026413 2004-07-26 2005-07-26 Traitement de troubles inflammatoires, auto-immunes ou autres a l'aide d'agents qui permettent de reduire la sequestration du zinc par la calprotectine WO2006014911A2 (fr)

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CN114062691A (zh) * 2022-01-12 2022-02-18 苏州和锐生物科技有限公司 一种稀释液以及钙卫蛋白校准品

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US20030099635A1 (en) * 2001-10-04 2003-05-29 Protein Therapeutics, Inc. Use of oral gammaglobulin for the treatment of immune-mediated diseases

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US20030099635A1 (en) * 2001-10-04 2003-05-29 Protein Therapeutics, Inc. Use of oral gammaglobulin for the treatment of immune-mediated diseases

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114062691A (zh) * 2022-01-12 2022-02-18 苏州和锐生物科技有限公司 一种稀释液以及钙卫蛋白校准品
CN114062691B (zh) * 2022-01-12 2022-04-01 苏州和锐生物科技有限公司 一种稀释液以及钙卫蛋白校准品

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