WO2013067415A1 - Identification et utilisation d'inhibiteurs de protéase pour traiter ou prévenir une drépanocytose - Google Patents

Identification et utilisation d'inhibiteurs de protéase pour traiter ou prévenir une drépanocytose Download PDF

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WO2013067415A1
WO2013067415A1 PCT/US2012/063404 US2012063404W WO2013067415A1 WO 2013067415 A1 WO2013067415 A1 WO 2013067415A1 US 2012063404 W US2012063404 W US 2012063404W WO 2013067415 A1 WO2013067415 A1 WO 2013067415A1
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calpain
erythrocytes
calcium
activity
mice
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Athar CHISHTI
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Tufts University
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/40—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against enzymes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies

Definitions

  • Sickle cell disease is a hereditary red cell disorder characterized by the presence of pathological hemoglobin (HbS), which polymerizes upon deoxygenation promoting red blood cell (RBC) dehydration and sickling.
  • HbS pathological hemoglobin
  • RBC red blood cell
  • Sickle cell disease patients require repeated hospitalizations, and it is estimated that such visits cost billions of dollars for annual hospitalizations in the US.
  • the treatment options for SCD have limited pharmacological tools for clinical practice. Hydroxyurea, with its limited effectiveness, is the only approved drug for SCD.
  • Other treatment options include repeated transfusions and bone marrow transplantation.
  • Calpains are cytosolic proteases found in all mammalian tissue and cell types. They are calcium-activated cysteine proteases, causing controlled proteolysis of protein substrates with regulatory functions. RBCs express only calpain-1 , whose physiological function remains poorly understood.
  • Calpains are widely expressed cysteine proteases activated by calcium at neutral pH.
  • Genome sequencing has identified at least 14 members of the calpain superfamily to date. Among them, calpain-1 and calpain-2, also designated as ⁇ -calpain and m-calpain respectively, remain the two most well-characterized members of the calpain-family of proteases.
  • Calpain-1 encoded by the Capnl gene
  • calpain-2 encoded by the Capn2 gene
  • calpain-2 is activated at a millimolar concentration of calcium in vitro.
  • calpain-1 calpain-2
  • calpastatin the endogenous inhibitor of both calpains, providing a regulatory mechanism for calcium-mediated activation of calpain activity.
  • Both calpain-1 and calpain-2 are expressed in varying levels in most mammalian tissues (Kawasaki, H. & Kawashima, S., Mol. Membr. Biol., 13:217-24, 1996).
  • calpain-1 dominates in the hematopoietic compartment whereas calpain-2 expression is more prominent in the non-hematopoietic cells. Since pharmacological inhibitors generally fail to distinguish between calpain-1 and calpain-2 activity, and may exert off-target effects, gene-targeting approaches have been used to tease out the individual function of each calpain in vivo.
  • the mature erythrocyte offers a unique model system where the physiological function of calpain-1 can be evaluated in the absence of calpain-2 activity. Moreover, the mature erythrocyte lacks any internal calcium storage organelles thus permitting assessment of calpain- 1 function at the plasma membrane.
  • Studies have demonstrated, for example, specific and limited calpain-mediated proteolysis of a membrane-bound calcium pump, also known as the plasma membrane Ca 2+ -ATPase or PMCA, which regulates calcium transport in mature erythrocytes (Papp, B. et al., J. Biol. Chem., 264:4577-82, 1989; Wang, K. et al., Adv. Exp. Med.
  • calpain-mediated proteolysis of hemoglobin provides a mechanism for the autocatalytic inactivation of calpain activity and formation of Heinz bodies (Pontremoli, S. et al., Biochem. Biophys. Res. Com., 123:331 -7, 1984).
  • Calpain activity plays a role in the regulation of protein phosphorylation of erythrocyte membrane proteins such as protein 4.1 R (Al, Z. & Cohen, C, Biochem. J., 296:675-83, 1993; Kishimoto, A. et al., J. Biol. Chem., 258:1 156-64, 1983).
  • calpain activity in erythrocytes has been traditionally investigated by employing the pharmacological inhibitors of cysteine proteases.
  • leupeptin and E-64 see Table I
  • calpeptin a calpain inhibitor that also inhibits the calpeptin-sensitive protein-tyrosine phosphatase that acts upstream of the RhoA GTPase (Schoenwaelder, S. & Burridge, K., J. Biol. Chem., 274:14359- 67, 1999).
  • RhoA GTPase RhoA GTPase
  • Calpain-1 loss leads to unexpected consequences with regard to erythrocyte shape, substrate cleavage, activity of transporters and phosphorylation of membrane proteins.
  • these studies demonstrate that selective pharmacological inhibition of calpain-1 offers new therapeutic options for erythrocyte pathologies with aberrant calcium homeostasis (e.g., sickle cell disease and/or ⁇ - thalassemia).
  • calpain-1 can be truncated and modified as a part of a fusion protein to inhibit calpain-1 in vivo (U.S. Patent No: 6,015,787), such inhibitors are difficult to administer, have traditionally more complicated production protocols, and can lead to pleiotropic effects. Described herein is the use of small-molecule inhibitors of calpain-1 with favorable pharmacokinetic properties that can be used to treat, for example, sickle cell disease and/or ⁇ -thalassemia.
  • the present disclosure is directed to a method of treating or preventing sickle cell disease, comprising: administering to a subject an effective amount of a protease inhibitor that inhibits a protease associated with sickle cell disease.
  • a protease inhibitor that inhibits a protease associated with sickle cell disease.
  • the protease associated with sickle cell disease is calpain-1 .
  • the present disclosure is directed to a composition for treating sickle cell disease, comprising a protease inhibitor that inhibits a protease associated with sickle cell disease.
  • the present disclosure is directed to a method of identifying a therapeutic agent for treating sickle cell disease, comprising contacting a cell or animal model of sickle cell disease with a candidate therapeutic agent, wherein observation of one or more sickle cell disease phenotypes that revert to a wild-type phenotype is indicative of the candidate agent's efficacy as a treatment for sickle cell disease.
  • the present disclosure is directed to a method of treating or preventing sickle cell disease or ⁇ -thalassemia, comprising: administering to a subject an effective amount of a small molecule or antibody that inhibits calpain-1 .
  • the protease inhibitor is a small molecule selected from the group consisting of the molecules of Table 1 .
  • the inhibitor is a small molecule that is derived from a molecule of Table I.
  • the small molecule is BDA-410 or a derivative thereof.
  • the method further comprises treating the subject with a therapeutically effective amount of hydroxyurea.
  • the present disclosure is directed to a composition for treating sickle cell disease or ⁇ -thalassemia, comprising a small molecule or antibody that inhibits calpain-1 .
  • the protease inhibitor is a small molecule selected from the group consisting of the molecules of Table 1 .
  • the inhibitor is a small molecule that is derived from a molecule of Table I.
  • the small molecule is BDA-410 or a derivative thereof.
  • the present disclosure is directed to a method of identifying a therapeutic agent for treating sickle cell disease or ⁇ -thalassemia, comprising contacting a cell or animal model of sickle cell disease with a candidate therapeutic agent, wherein observation of one or more sickle cell disease phenotypes that revert to a wild-type phenotype is indicative of the candidate agent's efficacy as a treatment for sickle cell disease.
  • the method further comprises measuring the efficacy of the candidate therapeutic agent for inhibiting calpain-1 protease activity.
  • the present disclosure is directed to the use for the manufacture of a medicament of a small molecule or antibody that inhibits calpain-1 for the treatment or prevention of sickle cell disease or ⁇ -thalassemia.
  • FIG. 1 shows blood smears after BDA-410 treatment.
  • BDA-410 treatment ameliorates sickle red cell morphology, reduces the dense red cell fraction in sickle cell SAD mice and increases sickle red cell K + content.
  • FIG. 1 A Representative pictures of red blood cells (RBCs) drawn from a wild-type and sickle cell SAD mouse at baseline and after 7 and 14 days (7d-14d) of treatment with BDA-410 at the dosage of 30 mg/Kg/day. The red cell pictures were captured with DM6000 Leica Microsystem (Germany), 63 ⁇ /0.8 dry objective.
  • FIG. 1 B Density profiles were obtained at baseline and after 7 and 14 days (7d-14d) of treatment with BDA-410 at the dosage of 30 mg/Kg/day.
  • FIG. 2 demonstrates human dense sickle red cells show reduced calpastatin activity.
  • FIG. 2A Inhibition of exogenous calpain-1 by heat stable extracts. Up to 30 ⁇ L of extract was added to the reaction mixture described in Example 3. Extracts were prepared from unfractionated normal red blood cells (unfx AA) ( ⁇ ), unfractionated sickle red blood cells (unfx SS) ( ⁇ ) and dense (>1 .10 g/cc, corresponding to fraction VI; fx VI SS) sickle red cells ( ⁇ ).
  • FIG. 1 unfractionated normal red blood cells
  • unfx SS unfractionated sickle red blood cells
  • dense >1 .10 g/cc, corresponding to fraction VI; fx VI SS
  • FIG. 2C Immunoblot analysis of heat stable extract showing decreased calpastatin protein in the dehydrated (fx VI) sickle RBC compared to unfractionated sickle red cells.
  • FIG. 3 demonstrates BDA-410 treatment reduces the activity of the Gardos channel and the amount of peroxiredoxin-2 associated to the membrane.
  • FIG. 3A Putative calpain specific cleavage sites on Ca 2+ -dependent K + channel. The putative domains are highlighted graphically, and the residues involved are listed above.
  • FIG. 3B Ca 2+ -activated K + channel (Gardos channel) activity in red cells from wild-type (WT) and sickle cell SAD mice at baseline and after 14 days treatment with BDA-410 at the dosage of 30 mg/Kg/day.
  • FIG. 3C Upper panel. Putative calpain specific cleavage sites on Prx2 are shown. The putative domains are highlighted graphically, and the residues involved are listed above. In Prx2 the position of the catalytic cysteine residues are reported (below). Lower panel.
  • FIG. 4 demonstrates BDA-410 treatment prevents the hypoxia-induced red cell dehydration and K + loss in sickle cell SAD mice.
  • FIG. 5 demonstrates proteolysis and phosphorylation of erythrocyte membrane proteins.
  • FIG. 5A SDS-PAGE of erythrocyte ghosts (Coomassie Blue: 50 ⁇ g total protein) isolated from WT (+/+) and calpain-1 null (-/-) mice. Erythrocytes were treated with calcium ionophore A23187 (1 .0 ⁇ ) and incubated with different concentrations of CaCI 2 at 37C for 10 minutes. The position of major membrane proteins is indicated.
  • FIG. 5B Western blot analysis of spectrin in WT (+/+) and calpain-1 null (-/-) ghosts at 400 and 1 ,000 ⁇ calcium.
  • FIG. 5C SDS-PAGE of erythrocyte ghosts (50 ⁇ g total protein) from WT (+/+) and calpain-1 null (-/-) mice. Intact erythrocytes were metabolically labeled ( 32 P) following stimulation with either 2.0 mM dibutyryl cAMP or 1 .0 ⁇ PMA, or 1 .0 ⁇ A23187 plus 500 ⁇ CaCI 2 for 30 minutes at 37C.
  • FIG. 5D Western blot analysis of protein kinase C (PKC) proteolysis in erythrocyte ghosts isolated from WT (+/+) and calpain-1 null (-/-) erythrocytes following activation either with 1 .0 ⁇ PMA or 1 .0 ⁇ A23187 plus 500 ⁇ CaCI 2 .
  • PKC protein kinase C
  • FIG. 6 demonstrates calcium-induced erythrocyte shape change.
  • FIG. 6A Erythrocytes from WT (+/+) and calpain-1 null (-/-) mice were treated with calcium ionophore A23187 (1 .5 ⁇ ) and incubated with 50 ⁇ CaCI 2 . After incubation at 37C for specified time intervals, erythrocyte shape change was quantified by phase contrast microscopy (bottom panels).
  • FIG. 6B Same conditions as shown in FIG. 6A except the concentration of calcium was increased to 1 .0 mM.
  • FIG. 7 shows erythrocyte deformability and filterability analysis.
  • FIG. 7A Quantification of erythrocyte deformability by Ektacytometry. Erythrocytes from WT and KO mice were resuspended in calcium-free Tyrode's buffer, and treated with calcium ionophore A23187 (1 .0 ⁇ ) and calcium (50 ⁇ ). Deformability measurements were performed in RheoScan D-300.
  • FIG. 7B Measurement of the erythrocyte filterability. Wild type (WT) and calpain-1 null (KO) erythrocytes were diluted to 0.1 % hematocrit, and allowed to filter through a 4.6 micron pore sized nickel mesh filter. Data shown are means ⁇ SD of 4-6 measurements from two
  • FIG. 8 shows erythrocyte osmotic fragility, density, and life span measurements.
  • FIG. 8A Washed erythrocytes from wild type (WT) and calpain-1 null (KO) mice were subjected to the osmotic fragility test.
  • FIG. 8B Osmotic fragility of WT and KO erythrocytes was performed using a narrow range of salt concentration.
  • FIG. 8C Density gradient separation of light (young) and dense (old) erythrocytes from WT and KO mice.
  • FIG. 8D Determination of erythrocyte life span in vivo. Flow cytometry was used to quantify the percentage of NHS-biotin- labeled erythrocytes as described in Example 4.
  • FIG. 9 shows measurement of K-CI cotransporter and calcium pump activity.
  • FIG. 10 shows atomic force microscopy (AFM) of mouse erythrocyte cytoskeleton.
  • Washed erythrocytes in phosphate buffered saline (PBS) were treated with calcium ionophore A23187 and 50 ⁇ calcium, attached onto the glass coverslips, lysed to expose the
  • the present disclosure relates to the discovery that the inhibition of regulatory proteases, e.g., calpain-1 , in red blood cells (RBCs), leads to the prevention and/or amelioration of sickle cell disease (SCD) or ⁇ -thalassemia.
  • SCD sickle cell disease
  • the correlation between calpain-1 activity and RBC morphology was observed in calpain-1 knockout mice, where the loss oc calpain-1 protected cells from changes to cytoskeleton instability (Wieschhaus, A. et ai, Biochem. J., 448:141 -52, 2012). This observation led to the present disclosure identifying the therapeutic effects of calpain-1 inhibitors.
  • Inhibition of calpain-1 inhibits the downstream regulatory effects of such regulatory proteases.
  • RBCs from a SCD animal or cellular model or subject afflicted with SCD exhibit sickling and dehydration
  • the effects of inhibiting regulatory proteases can be, for example, assessed by observing the phenotypic morphological changes to RBCs upon treatment with inhibitors.
  • the present disclosure uses small molecule calpain-1 inhibitor(s), e.g., BDA-410 and derivatives thereof, that inhibit the ability of calpain-1 to regulate downstream effectors of SCD.
  • the present disclosure therefore, describes the use of regulatory protease inhibitors, e.g., inhibitors of calpain-1 , e.g., small molecule inhibitors contained in Table I, for example, or antibody inhibitors, for preventing or treating SCD or ⁇ -thalassemia. Also described herein are methods for identifying therapeutic agents useful for treating of preventing SCD or ⁇ -thalassemia. The present disclosure also describes therapeutic targets that are downstream targets of calpain-1 . These calpain-1 targets can be regulated to treat or prevent SCD or ⁇ -thalassemia.
  • SCD is a hereditary red cell disorder characterized by the presence of pathological HbS, which polymerizes upon deoxygenation promoting red blood cell (RBC) dehydration and sickling.
  • RBC red blood cell
  • Dense, dehydrated sickle RBCs play a crucial role in the pathogenesis of clinical manifestation as well as in acute and chronic organ damage in SCD.
  • Studies on sickle RBC membrane permeability have identified two membrane transport systems involved in generation of dense sickle RBCs: the K-CI cotransport and the Ca 2+ -activated-K + channel (Gardos channel).
  • Calpains are calcium dependent cysteine proteinases that selectively cleave proteins in response to calcium. Human RBCs express only calpain-1 and calpastatin, its endogenous inhibitor.
  • RBC membrane and cytoskeleton proteins are targets of calpain, which, by causing limited degradation, regulates the activities of membrane-associated enzymes and proteins.
  • Calpain-1 activates the Ca 2+ ATPase pump. Exposure of RBCs to peroxynitrite induces activation of calpain-1 (Boivin, P. et ai, Int. J. Biochem., 22:1479-89, 1990; Ciana, A. et ai, Bioelectrochemistry, 62:169-73, 2004; Azam, M. et ai., Mol. Cell. Biol., 21 :2213-20, 2001 ;
  • Calpain-1 expression is similar in normal and sickle RBCs. Calpain-1 activation of the
  • Ca 2+ pump is greater in sickle than in control RBCs. Calpastatin expression and activity are decreased in dehydrated sickle RBCs (Olorunsogo, O. et ai., Biosci. Rep., 10:281 -91 , 1990).
  • Inhibitors of calpain-1 can include, for example, the macromolecular natural inhibitor, calpastatin, small molecule inhibitors (e.g., those described in Table I), and antibodies.
  • BDA- 410 and derivatives thereof, for example, are orally active small molecules that are selective inhibitors of calpain-1 . They have been used to investigate diseases such as Alzheimer's disease and malaria (Battaglia, F. et ai., J. Mol. Neurosci., 20:357-62, 2003; Li, X. et ai., Mol. Biochem. ParasitoL, 155:26-32, 2007; Trinchese, F. et ai, J. Clin. Invest., 1 18:2796-807, 2008 ; also see U.S. patent Nos : 5,328,909 ; 5,395,958 and 5,416,1 17, the entire contents of each of which is hereby incorporated by reference).
  • calpain- 1 Described herein are data showing the relevance of gene inactivation of mouse calpain- 1 , which revealed differential regulation of RBC calcium pump, enhanced hydration, and filterability properties, for SCD.
  • BDA-410 an orally active inhibitor of calpain-1 . was used (Battaglia, F. et al., J. Mol. Neurosci., 20:357-62, 2003; Trinchese, F. et al., J. Clin. Invest., 1 18:2796-807, 2008; Li, X. et al., Mol. Biochem.
  • BDA-410 was administrated to wild-type (WT) and sickle cell (SAD) mice.
  • WT wild-type
  • SAD sickle cell
  • BDA-410 treatment reduced Gardos channel activity and prevented the hypoxia-induced potassium loss and red cell dehydration in SAD mice.
  • pharmacological inhibition of calpain-1 by BDA-410 or similar inhibitors either alone or in combination of existing anti-sickling compounds such as hydroxyurea offers a new therapeutic strategy to reduce red cell dehydration and associated pathologies in SCD.
  • module refers to a change in the quality or quantity of a gene, protein or any molecule that is inside, outside, or on the surface of a cell.
  • the change can be an increase or decrease in expression or level of the molecule.
  • modulates also includes changing the quality or quantity of a biological function or enzymatic activity.
  • the term "modulator” refers to a composition that modulates one or more physiological or biochemical events.
  • the modulator inhibits one or more biological activities associated with, for example, a regulatory protease associated with SCD.
  • the modulator is a small molecule, an antibody, a mimetic, a decoy or an oligonucleotide.
  • the modulator acts by blocking ligand binding or by competing for a ligand-binding site.
  • the modulator acts independently of ligand binding.
  • the modulator does not compete for a ligand binding site.
  • small molecule or “small molecule therapeutic” refers to a low molecular weight organic compound that is not a polymer.
  • the upper molecular weight limit for a small molecule is approximately 800 Daltons, which allows for the possibility of small molecules to rapidly diffuse across cell membranes so that they can reach intracellular sites of action. This molecular weight cutoff also allows for oral bioavailability for many small molecules.
  • regulatory protease inhibitors can inhibit one or more regulatory proteases associated with SCD, e.g., calpain-1 . Inhibition can be at least 25%, at least 50%, at least 75%, at least 80%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100%, as compared to a control.
  • BDA-410 has a K, for calpain-1 of 130nM in vitro and an IC 50 of 21 .4nM in cell assays (SHSY5Y cells). It is otherwise non-toxic as determined by bacterial reverse mutation assays, Drosophila somatic mutation and recombination tests, inhibition of metabolic cooperation assays and replicative DNA synthesis assays. Toxicity in rats for oral administration is >2000mg/kg.
  • a dosage of 100mg/kg administered in a volume of 5mL/kg(po) in a 1 % Tween80 saline suspension is sufficient to inhibit calain-1 (30mg/kg).
  • a 100 mg/kg dose can be prepared, for example, by pulverizing 60mg of BDA-410 and resuspending the pulverized powder in 30 ⁇ L of Tween80. Saline solution is then used to reach a final volume of 3ml_.
  • treatment refers to the administration of a therapeutic agent or the performance of medical procedures with respect to a patient or subject, for either prophylaxis (prevention) or to cure or reduce the symptoms of the infirmity or malady in the instance where the patient is afflicted.
  • prevention of SCD or SCD symptoms is included within the scope of treatment.
  • the methods and compounds described herein or identified through methods described herein can be used as part of a treatment regimen in therapeutically effective amounts, as would be determined by one of skill in the art.
  • a "therapeutically effective amount” is an amount sufficient to decrease, prevent or ameliorate the symptoms associated with a medical condition, e.g., SCD or SCD symptoms.
  • the present disclosure for example, is directed to treatment using a therapeutically effective amount of a compound sufficient to treat SCD or SCD symptoms.
  • the terms "individual,” “subject,” “host” and “patient” are used interchangeably and refer to any subject for whom diagnosis, treatment, or therapy is desired, particularly humans. Other subjects can include other mammals, for example, cattle, dogs, cats, guinea pigs, rabbits, rats, mice, horses, and the like.
  • the treatment(s) described herein are understood to utilize formulations including compounds identified herein or identified through methods described herein and, for example, salts, solvates and co-crystals of the compound(s).
  • the compounds of the present disclosure can exist in unsolvated as well as solvated forms with pharmaceutically acceptable solvents such as, for example, water, ethanol, and the like. In general, the solvated forms are considered equivalent to the unsolvated forms for the purposes of the present disclosure.
  • pharmaceutically acceptable salts, esters, amides and prodrugs refers to those carboxylate salts, amino acid addition salts, esters, amides, prodrugs and inclusion complexes of the compounds of the present disclosure that are, within the scope of sound medical judgment as determined by one of skill in the art, suitable for use in contact with the tissues of patients without undue toxicity, irritation, allergic response, and the like, commensurate with a reasonable benefit/risk ratio, and effective for their intended use, as well as the zwitterionic forms, where possible, of the compounds of the disclosure.
  • prodrug refers to compounds that are rapidly transformed in vivo to yield the parent compounds of the above formula, for example, by hydrolysis in blood (T. Higuchi and V. Stella, "Pro-drugs as Novel Delivery Systems,” Vol. 14 of the A.C.S. Symposium Series;
  • Activation in vivo can come about, for example, by chemical action or through the intermediacy of enzymes. Microflora in the Gl tract can also contribute to activation in vivo.
  • solvate refers to a compound in the solid state, wherein molecules of a suitable solvent are incorporated.
  • a suitable solvent for therapeutic administration is
  • solvates are formed by dissolving the compound in the appropriate solvent and isolating the solvate by cooling or using an antisolvent.
  • the solvate is typically dried or azeotroped under ambient conditions.
  • Co-crystals are combinations of two or more distinct molecules arranged to create a unique crystal form whose physical properties are different from those of its pure constituents (Remenar, J. et al., J. Am. Chem. Soc, 125:8456- 8457, 2003). Inclusion complexes are described in Remington: The Science and Practice of Pharmacy 19.sup.th Ed.
  • the compounds can be presented as salts.
  • pharmaceutically acceptable salt refers to salts whose counter ion derives from pharmaceutically acceptable non-toxic acids and bases.
  • Suitable pharmaceutically acceptable base addition salts for the compounds of the present disclosure include, but are not limited to, metallic salts made from aluminum, calcium, lithium, magnesium, potassium, sodium and zinc or organic salts made from lysine, N,N-dialkyl amino acid derivatives (e.g., ⁇ , ⁇ -dimethylglycine, piperidine-1 -acetic acid and morpholine-4- acetic acid), ⁇ , ⁇ '-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine, ethylenediamine, meglumine (N-methylglucamine) and procaine.
  • metallic salts made from aluminum, calcium, lithium, magnesium, potassium, sodium and zinc or organic salts made from lysine, N,N-dialkyl amino acid derivatives (e.g., ⁇ , ⁇ -dimethylgly
  • suitable pharmaceutically acceptable base addition salts for the compounds include, for example, inorganic acids and organic acids. Examples include acetate, benzenesulfonate (besylate), benzoate, bicarbonate, bisulfate, carbonate, camphorsulfonate, citrate, ethanesulfonate, fumarate, gluconate, glutamate, bromide, chloride, isethionate, lactate, maleate, malate, mandelate, methanesulfonate, mucate, nitrate, pamoate, pantothenate, phosphate, succinate, sulfate, tartrate, p-toluenesulfonate, and the like (Barge, S. et al., J.
  • Diluents that are suitable for use in the pharmaceutical composition of the present disclosure include, for example, pharmaceutically acceptable inert fillers such as
  • microcrystalline cellulose lactose, sucrose, fructose, glucose dextrose, or other sugars, dibasic calcium phosphate, calcium sulfate, cellulose, ethylcellulose, cellulose derivatives, kaolin, mannitol, lactitol, maltitol, xylitol, sorbitol, or other sugar alcohols, dry starch, saccharides, dextrin, maltodextrin or other polysaccharides, inositol or mixtures thereof.
  • the diluent can be, for example, a water-soluble diluent. Examples of preferred diluents include, for example:
  • microcrystalline cellulose such as Avicel PH1 12, Avicel PH101 and Avicel PH102 available from FMC Corporation; lactose such as lactose monohydrate, lactose anhydrous, and Pharmatose DCL 21 ; dibasic calcium phosphate such as Emcompress; mannitol; starch; sorbitol; sucrose; and glucose.
  • Diluents are carefully selected to match the specific composition with attention paid to the compression properties.
  • the diluent can be used in an amount of about 2% to about 80% by weight, about 20% to about 50% by weight, or about 25% by weight of the treatment formulation.
  • agents that can be used in the treatment formulation include, for example, a surfactant, dissolution agent and/or other solubilizing material.
  • Surfactants that are suitable for use in the pharmaceutical composition of the present disclosure include, for example, sodium lauryl sulphate, polyethylene stearates, polyethylene sorbitan fatty acid esters, polyoxyethylene castor oil derivatives, polyoxyethylene alkyl ethers, benzyl benzoate, cetrimide, cetyl alcohol, docusate sodium, glyceryl monooleate, glyceryl monostearate, glyceryl palmitostearate, lecithin, medium chain triglycerides, monoethanolamine, oleic acid, poloxamers, polyvinyl alcohol and sorbitan fatty acid esters.
  • Dissolution agents increase the dissolution rate of the active agent and function by increasing the solubility of the active agent.
  • Suitable dissolution agents include, for example, organic acids such as citric acid, fumaric acid, tartaric acid, succinic acid, ascorbic acid, acetic acid, malic acid, glutaric acid and adipic acid, which may be used alone or in combination. These agents can also be combined with salts of the acids, e.g., sodium citrate with citric acid, to produce a buffer system.
  • Other agents that can be used to alter the pH of the microenvironment on dissolution include salts of inorganic acids and magnesium hydroxide.
  • Disintegrants that are suitable for use in the pharmaceutical composition of the present disclosure include, for example, starches, sodium starch glycolate, crospovidone,
  • croscarmellose microcrystalline cellulose, low substituted hydroxypropyl cellulose, pectins, potassium methacrylate-divinylbenzene copolymer, polyvinyl alcohol), thylamide, sodium bicarbonate, sodium carbonate, starch derivatives, dextrin, beta cyclodextrin, dextrin derivatives, magnesium oxide, clays, bentonite and mixtures thereof.
  • the active ingredient of the present disclosure can be mixed with excipients that are pharmaceutically acceptable and compatible with the active ingredient and in amounts suitable for use in the therapeutic methods described herein.
  • excipients can be homogeneously mixed with the active agent of the present disclosure as would be known to those skilled in the art.
  • the active agent for example, can be mixed or combined with excipients such as but not limited to microcrystalline cellulose, colloidal silicon dioxide, lactose, starch, sorbitol, cyclodextrin and combinations of these.
  • compositions of the present disclosure can also optionally include other therapeutic ingredients, anti-caking agents, preservatives, sweetening agents, colorants, flavors, desiccants, plasticizers, dyes, and the like.
  • compositions are administered in combination with a second therapeutic agent.
  • any such optional ingredient must, of course, be compatible with the compound of the disclosure to insure the stability of the formulation.
  • the dose range for adult humans is generally from 0.1 ⁇ g to 10 g/day orally. Tablets or other forms of presentation provided in discrete units can conveniently contain an amount of compound of the disclosure that is effective at such dosage or as a multiple of the same, for instance, units containing 0.1 mg to 500 mg, usually around 5 mg to 200 mg.
  • the precise amount of compound administered to a patient will be the responsibility of the attendant physician.
  • the dose employed will depend on a number of factors, including, for example, the age and sex of the patient, the precise disorder being treated, and its severity. The frequency of administration depends on the
  • pharmacodynamics of the individual compound and the formulation of the dosage form which is optimized by methods known in the art (e.g., controlled or extended release tablets, enteric coating etc.).
  • the compounds disclosed herein are optionally substituted with one or more substituents.
  • substituents within this context include, for example, halogen, hydroxy, alkyl, alkoxy, alkanoyl, nitro, cyano, oxo, carbocyclyl,
  • Ra and Rb in this context may be the same or different and independently hydrogen, halogen, hydroxyl, alkyl, alkoxy, alkanoyl, amino, alkylamino, dialkylamino, alkylthiol, carbocyclyl, carbocycloalkyl, heterocarbocyclyl,
  • heterocarbocycloalkyl aryl, arylalkyl, heteroaryl, heteroarylalkyl.
  • optionally substituted means that substitution is optional and therefore it is possible for the designated atom or compound is unsubstituted.
  • a substituted molecule is "derived" from the unsubstituted or
  • derivatives of, for example, BDA-410 can include the following:
  • R 1 is selected from H, C 1 -C 12 alkyl, C 3 -C 12 cycloalkyl, C 3 -C 12 heterocyclic, aryl, heteroaryl, R 12 SO 2 -, R 12 C(O)-, R 12 OC(O)-, R 12 R 13 NC(O)-, optionally substituted with one or more R 14 ;
  • R 2 , R 4 is independently selected from H, C 1 -C 12 alkyl, C 3 -C 12 cycloalkyl, C 3 -C 12 heterocyclic, optionally substituted with one or more R 14 ;
  • R 3 , R 5 and R 8 is independently selected from H, C 1 -C 12 alkyl, C 3 -C 12 cycloalkyl, C 3 -C 12 heterocyclic, aryl, heteroaryl, optionally substituted with one or more R 14 ;
  • R 6 , R 7 is independently selected from H, halogen, OH, oxo, -OR 12 , -NR 12 R 13 , C 1 -C 12 alkyl, C 3 -C 12 cycloalkyl, optionally substituted with one or more R 14 ;
  • R 12 and R 13 is independently selected from H, C 1 -C 12 alkyl, C 3 -C 12 cycloalkyl, C 3 -C 12
  • R 14 is independently selected from: halogen, -OR 12 , -NO 2 , CN, CF 3 , OCF 3 , R 12 , oxo, thioxo, 1 ,2-methylenedioxy, 1 ,2-ethylenedioxy, NR 12 R 13 , SR 12 , SOR 12 , SO 2 R 12 , SO 2 NR 12 R 13 , S0 3 R 12 , C(O)R 12 , C(O)C(O)R 12 , C(O)CH 2 C(O)R 12 , C(S)R 12 , C(S)OR 12 , C(O)OR 12 , C(O)OR 12 , C(O)C(O)OR 12 , C(O)C(O)OR 12 , C(O)C(O)NR 12 R 13 , OC(O)R 12 , C(O)NR 12 R 13 , OC(O)NR 12 R 13 , C(S)NR 12 R 13 , (CH
  • R 1 is selected from H, C 1 -C 12 alkyl, C 3 -C 12 cycloalkyl, C 3 -C 12 heterocyclic, aryl, heteroaryl, R 12 SO 2 -, R 12 C(O)-, R 12 OC(O)-, R 12 R 13 NC(O)-, optionally substituted with one or more R 14 ;
  • R 2 , R 4 is independently selected from H, C 1 -C 12 alkyl, C 3 -C 12 cycloalkyl, C 3 -C 12 heterocyclic, optionally substituted with one or more R 14 ;
  • R 3 , R 5 , R 8 , R 9 and R 10 is independently selected from H, C 1 -C 12 alkyl, C 3 -C 12 cycloalkyl, C 3 -C 12 heterocyclic, aryl, heteroaryl, optionally substituted with one or more R 14 ;
  • R 6 , R 7 is independently selected from H, halogen, OH, oxo, -OR 12 , -NR 12 R 13 , C 1 -C 12 alkyl, C 3 -C 12 cycloalkyl, optionally substituted with one or more R 14 ;
  • R 12 and R 13 is independently selected from H, C 1 -C 12 alkyl, C 3 -C 12 cycloalkyl, C 3 -C 12
  • R 14 is independently selected from:
  • R 1 is selected from H, C 1 -C 12 alkyl, C 3 -C 12 cycloalkyl, C 3 -C 12 heterocyclic, aryl, heteroaryl, R 12 SO 2 -, R 12 C(O)-, R 12 OC(O)-, R 12 R 13 NC(O)-, optionally substituted with one or more R 14 ;
  • R 2 , R 4 is independently selected from H, C 1 -C 12 alkyl, C 3 -C 12 cycloalkyl, C 3 -C 12 heterocyclic, optionally substituted with one or more R 14 ;
  • R 3 , R 5 , R 8 , R 9 and R 10 is independently selected from H, C 1 -C 12 alkyl, C 3 -C 12 cycloalkyl, C 3 -C 12 heterocyclic, aryl, heteroaryl, optionally substituted with one or more R 14 ;
  • R 6 , R 7 is independently selected from H, halogen, OH, oxo, -OR 12 , -NR 12 R 13 , C 1 -C 12 alkyl, C 3 -C 12 cycloalkyl, optionally substituted with one or more R 14 ;
  • R 12 and R 13 is independently selected from H, C 1 -C 12 alkyl, C 3 -C 12 cycloalkyl, C 3 -C 12
  • R 14 is independently selected from: halogen, -OR 12 , -NO 2 , CN, CF 3 , OCF 3 , R 1 2, oxo, thioxo, 1 ,2-methylenedioxy, 1 ,2-ethylenedioxy, NR 12 R 13 , SR 12 , SOR 12 , SO 2 R 12 , SO 2 NR 12 R 13 , S0 3 R 12 , C(O)R 12 , C(O)C(O)R 12 , C(O)CH 2 C(O)R 12 , C(S)R 12 , C(S)OR 12 , C(O)OR 12 , C(O)OR 12 , C(O)C(O)OR 12 , C(O)C(O)OR 12 , C(O)C(O)NR 12 R 13 , OC(O)R 12 , C(O)NR 12 R 13 , OC(O)NR 12 R 13 , C(S)NR 12 R 13 , (CH 2
  • R 1 is selected from H, C 1 -C 12 alkyl, C 3 -C 12 cycloalkyl, C 3 -C 12 heterocyclic, aryl, heteroaryl, R 12 SO 2 -, R 12 C(O)-, R 12 OC(O)-, R 12 R 13 NC(O)-, optionally substituted with one or more R 14 ;
  • R 2 , R 4 is independently selected from H, C 1 -C 12 alkyl, C 3 -C 12 cycloalkyl, C 3 -C 12 heterocyclic, optionally substituted with one or more R14;
  • R 3 , R 5 , R 8 , R 9 , R 10 , R 1 and R 15 are independently selected from H, C 1 -C 12 alkyl, C 3 -C 12 cycloalkyl, C 3 -C 12 heterocyclic, aryl, heteroaryl, optionally substituted with one or more R 14 ;
  • R 6 , R 7 is independently selected from H, halogen, OH, oxo, -OR 12 , -NR 12 R 13 , C 1 -C 12 alkyl, C 3 -C 12 cycloalkyl, optionally substituted with one or more R 14 ;
  • R 12 and R 13 is independently selected from H, C 1 -C 12 alkyl, C 3 -C 12 cycloalkyl, C 3 -C 12
  • R 14 is independently selected from: halogen, -OR 12 , -NO 2 , CN, CF 3 , OCF 3 , R 12 , oxo, thioxo, 1 ,2-methylenedioxy, 1 ,2-ethylenedioxy, NR 12 R 13 , SR 12 , SOR 12 , SO 2 R 12 , SO 2 NR 12 R 13 , SO 3 R 12 , C(O)R 12 , C(O)C(O)R 12 , C(O)CH 2 C(O)R 12 , C(S)R 12 , C(S)OR 12 , C(O)OR 12 , C(O)OR 12 , C(O)C(O)OR 12 , C(O)C(O)OR 12 , C(O)C(O)NR 12 R 13 , OC(O)R 12 , C(O)NR 12 R 13 , OC(O)NR 12 R 13 , C(S)NR 12 R 13 , (CH 2
  • alkyl means a noncyclic straight chain or branched, unsaturated or saturated hydrocarbon such as those containing from 1 to 10 carbon atoms, while the term “lower alkyl” or “C 1-6 alkyl” has the same meaning as alkyl but contains from 1 to 6 carbon atoms. The term “higher alkyl” has the same meaning as alkyl but contains from 7 to 10 carbon atoms.
  • saturated straight chain alkyls include, for example, methyl, ethyl, n- propyl, n-butyl, n-pentyl, n-hexyl, n-septyl, n-octyl, n-nonyl, and the like; while saturated branched alkyls include isopropyl, sec-butyl, isobutyl, tert-butyl, isopentyl, and the like.
  • Unsaturated alkyls contain at least one double or triple bond between adjacent carbon atoms (referred to as an "alkenyl” or "alkynyl,” respectively).
  • Representative straight chain and branched alkenyls include, for example, ethylenyl, propylenyl, 1 -butenyl, 2-butenyl, isobutylenyl, 1 -pentenyl, 2-pentenyl, 3 -methyl- 1 -butenyl, 2-methyl-2-butenyl, 2,3- dimethyl-2-butenyl, and the like; while representative straight chain and branched alkynyls include acetylenyl, propynyl, 1 -butynyl, 2-butynyl, 1 -pentynyl, 2-pentynyl, 3- methyl-1 -butynyl, and the like.
  • haloalkyl refers to halo-substituted alkyl groups.
  • Non-aromatic mono or polycyclic alkyls are referred to herein as "carbocycles" or “carbocyclyl” or “cyclic alkyl” groups.
  • Representative saturated carbocycles include, for example, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and the like; while unsaturated carbocycles include, for example, cyclopentenyl and cyclohexenyl, aryls and the like.
  • Heterocarbocycles or “heterocarbocyclyl” groups are carbocycles that contain from one to four heteroatoms independently selected from, for example, nitrogen, oxygen and sulfur (which may be saturated or unsaturated (but not aromatic)), monocyclic or polycyclic, and wherein the nitrogen and sulfur heteroatoms can be optionally oxidized, and the nitrogen heteroatom can be optionally quaternized.
  • Heterocarbocycles include, for example,
  • morpholinyl pyrrolidinonyl, pyrrolidinyl, piperidinyl, hydantoinyl, valerolactamyl, oxiranyl, oxetanyl, tetrahydrofuranyl, tetrahydropyranyl, tetrahydropyridinyl, tetrahydroprimidinyl, tetrahydrothiophenyl, tetrahydrothiopyranyl, tetrahydropyrimidinyl, tetrahydrothiophenyl, tetrahydrothiopyranyl, and the like.
  • Aryl means an aromatic carbocyclic monocyclic or polycyclic ring such as phenyl or naphthyl.
  • heteroaryl refers an aromatic heterocarbocycle having one to four heteroatoms selected from, for example, nitrogen, oxygen and sulfur, and containing at least one carbon atom, including both mono- and polycyclic ring systems.
  • Polycyclic ring systems can, but are not required to, contain one or more non-aromatic rings, as long as one of the rings is aromatic.
  • heteroaryls are, for example, furyl, benzofuranyl, thiophenyl, benzothiophenyl, pyrrolyl, indolyl, isoindolyl, azaindolyl, pyridyl, quinolinyl, isoquinolinyl, oxazolyl, isooxazolyl, benzoxazolyl, pyrazolyl, imidazolyl, benzimidazolyl, thiazolyl,
  • heteroaryl includes, for example, N-alkylated derivatives such as a 1 -methylimidazol- 5-yl substituent.
  • heterocycle or “heterocyclyl” refers to mono- and polycyclic ring systems having one to four heteroatoms selected from, for example, nitrogen, oxygen and sulfur, and containing at least one carbon atom.
  • the mono- and polycyclic ring systems can be aromatic, non-aromatic or mixtures of aromatic and non-aromatic rings.
  • Heterocycle includes heterocarbocycles, heteroaryls, and the like.
  • Alkoxy refers to an alkyl group as defined above with the indicated number of carbon atoms attached through an oxygen bridge. Examples of alkoxy include, but are not limited to, methoxy, ethoxy, n-propoxy, i-propoxy, n-butoxy, s-butoxy, t-butoxy, n- pentoxy, and s-pentoxy.
  • Alkylamino refers an alkyl group as defined above with the indicated number of carbon atoms attached through an amino bridge.
  • An example of an alkylamino is methylamino, (e.g., - NH-CH 3 ).
  • the compounds of this disclosure can exist in radiolabeled form, i.e., the compounds can contain one or more atoms containing an atomic mass or mass number different from the atomic mass or mass number most commonly found in nature.
  • Radioisotopes of, for example, hydrogen, carbon, phosphorous, fluorine, and chlorine include 2 H, 3 H, 3 C, 4 C, 5 N, 35 S, 8 F and 36 CI, respectively.
  • Compounds that contain those radioisotopes and/or other radioisotopes of other atoms are within the scope of this disclosure.
  • Radiolabeled compounds of the present disclosure and prodrugs thereof can generally be prepared by methods known to those skilled in the art.
  • the compounds described herein can contain asymmetric centers and can thus give rise to enantiomers, diastereomers and other stereoisomeric forms.
  • Each chiral center can be defined in terms of absolute stereochemistry as (R)- or (S)-.
  • the present disclosure is meant to include all such possible isomers, as well as, their racemic and optically pure forms.
  • Optically active (R)- and (S)-isomers can be prepared using chiral synthons or chiral reagents, or resolved using conventional techniques.
  • the representation of the configuration of any carbon- carbon double bond appearing herein is selected for convenience only, and unless explicitly stated, is not intended to designate a particular configuration. Thus a carbon-carbon double bond depicted arbitrarily as E can be Z, E, or a mixture of the two in any proportion. Likewise, all tautomeric forms are also intended to be included.
  • the formulations include those suitable for oral, parenteral (including subcutaneous, intradermal, intramuscular, intravenous and intraarticular), rectal and topical (including dermal, buccal, sublingual and intraocular) administration.
  • the most suitable route depends upon the condition and disorder of the recipient.
  • the formulations can conveniently be presented in unit dosage form and can be prepared by any of the methods known in the art of pharmacy. All methods include the step of bringing into association at least one compound of the present disclosure or a pharmaceutically acceptable salt or solvate thereof ("active ingredient”) with the carrier, which constitutes one or more accessory ingredients.
  • active ingredient a pharmaceutically acceptable salt or solvate thereof
  • the formulations are prepared by uniformly and intimately bringing into association the active ingredient with liquid carriers or finely divided solid carriers or both and then, if necessary, shaping the product into the desired formulation.
  • the pharmaceutical preparations of the disclosure can be provided in a unit dosage form, and can be suitably packaged, for example in a box, blister, vial, bottle, sachet, ampoule or in any other suitable single-dose or multi-dose holder or container (which may be properly labeled); optionally with one or more leaflets containing product information and/or instructions for use.
  • Such unit dosages generally contain between 1 and 1000 mg, and usually between 5 and 500 mg, of the at least one compound of the disclosure, e.g., about 10, 25, 50, 100, 200, 300 or 400 mg per unit dosage.
  • Formulations of the present disclosure suitable for oral administration can be presented as discrete units such as capsules, cachets or tablets each containing a predetermined amount of the active ingredient; as a powder (including micronized and nanoparticulate powders) or granules; as a solution or a suspension in an aqueous liquid or a non-aqueous liquid; or as an oil-in-water liquid emulsion or a water-in-oil liquid emulsion.
  • the active ingredient can also be presented as a bolus, electuary or paste.
  • Combination therapy can be achieved by administering two or more agents, each of which is formulated and administered separately, or by administering two or more agents in a single formulation.
  • the second active ingredient can be, for example, a second compound identified herein or through screens described herein, or active ingredients useful for treating, for example, SCD or SCD symptoms, or symptoms associated with treatment by the first active agent ("side effects").
  • side effects active ingredients useful for treating, for example, SCD or SCD symptoms, or symptoms associated with treatment by the first active agent ("side effects").
  • side effects active ingredients useful for treating, for example, SCD or SCD symptoms, or symptoms associated with treatment by the first active agent
  • side effects side effects
  • Other combinations are also encompassed by combination therapy.
  • two agents can be formulated together and administered in conjunction with a separate formulation containing a third agent. While the two or more agents in the combination therapy can be administered simultaneously, they need not be.
  • administration of a first agent can precede administration of a second agent (or combination of agents) by minutes, hours, days, or weeks.
  • the two or more agents can be administered within minutes of each other or within any number of hours of each other or within any number or days or weeks of each other.
  • kits for treating or preventing SCD and/or ⁇ - thalassemia comprising one or more compounds described herein or identified through the screening methods provided herein.
  • the kits of the present disclosure can include, for example, components necessary for delivering a therapeutically effective amount of the active agent, instructions for use and/or devices for delivery of the active agent(s).
  • the present disclosure is also directed to models and screening methods useful for identifying molecules that inhibit calpain-1 and that are useful for treating SCD and/or ⁇ - thalassemia.
  • Compounds as identified can be used in the methods, formulations and kits described herein.
  • the compounds identified herein can be validated for their efficacy in treating SCD and/or ⁇ -thalassemia for example, by using cellular, animal and/or human models as outlined herein and in the "Exemplification.”
  • a candidate test compound which can include a compound designed based on the structure of, for example, a small molecule inhibitor of calain-1 , is assayed for its cytotoxic activity and/or its ability to inhibit calpain-1 and/or its ability to alter RBC morphology.
  • BDA-410 a novel orally active inhibitor of calpain-1 .
  • SAD sickle mouse
  • WT wild-type
  • SAD sickle cell
  • mice at baseline, day 7 and day 14 of BDA-410 treatment were evaluated for hematological parameters including the RBC density profile with phthalate density curves, RBC cation content, and Ca 2+ activated K + channel (Gardos channel) activity.
  • BDA-410 induced a significant increase in Hct in both WT and SAD mice with no significant changes in Hb levels and an associated increased in MCV.
  • the red cell K + content increased significantly in SAD RBCs at day 7 and day 14 of inhibitor treatment as compared to untreated SAD mice; whereas no major changes were observed in the WT RBCs.
  • the mean corpuscular Hb concentration (CHCM) decreased in both WT and SAD mice treated with BDA-410.
  • mice genetically lacking calpain-1 (calp-1 -/- ) generated by targeted gene inactivation were used.
  • Transgenic Hbb single/single SAD1 (SAD) mice (between 3-6 months of age, weight 25- 30 gr) were used as mouse model for sickle cell disease and matched C57B6/2J mice were used as controls (wild-type mice, WT).
  • Mice from both strains were divided into 4 groups of 6 mice each: two groups from each strain were treated with BDA-410 at the dosage of 30 mg/K/d by gavage once a day for 14 days while the others were exposed to the delivery vehicle only. The following parameters were evaluated at baseline and after 7 and 14 days of treatment: hematological parameters, red cell density profile, cation content, red cell morphology and Ca 2+ activated-K + channel activity.
  • mice were sacrificed for red cell membrane ghost preparation for immunoblot analysis and measurement of the amount of carbonyl groups.
  • mice from both strains were treated with BDA- 410 at the dosage of 30 mg/K/d by gavage for 14 days once a day and exposed to hypoxia 8% oxygen for 48 hours followed by 2 hours reoxygenation (De Franceschi, L. et al.,
  • Red cells genetically lacking calpain-1 show reduced Ca 2+ ATPase pump activity, which is restored by exogenous calmodulin.
  • the calpain-1 inhibitor BDA-410 induced amelioration of red cell morphology, increased Hct and MCV, decreased MCHC with reduction in dense red cells and increase RBC K + content, decreased Gardos channel activity and decreased membrane association of Prx2.
  • BDA-410 reduced the hypoxia-induced red cell K + loss and dehydration.
  • BDA-410-mediated calpain- 1 inhibition may represent a novel therapeutic approach in SCD.
  • SCD is a worldwide distributed hereditary red blood cell (RBC) disorder.
  • RBC hereditary red blood cell
  • One of the hallmarks of SCD is the presence of circulating dense RBCs, which play a key role in SCD- related clinical manifestations.
  • dense sickle-RBCs reduced calpastatin activity and protein expression were observed compared to either healthy RBCs or unfractionated sickle-RBCs, suggesting an imbalance between activator/inhibitor of calpain-1 in favor of activator in dense sickle-RBCs.
  • Calpain-1 is a non-lysosomal-cysteine -proteinase that modulates multiple cell functions through the selective cleavage of proteins.
  • calphostatin-C a specific inhibitor of protein-kinase -C (PKC)
  • PKC protein-kinase -C
  • Red cell suspensions containing 100 ⁇ L of cells were washed three times with 150 mM NaCl and after complete removal of the supernatant were lysed by adding 900 ⁇ L of lysis buffer (10 mM Tris-HCl , 0.5 mM DTT, 1 mM EDTA, 1 mM EGTA) followed by two fast freeze/thaw cycles using dry ice/alcohol. The freeze/thaw cycles help assure complete lysis of the dehydrated population of sickle cells. The lysate was spun at high speed to pellet membranes and the supernatant isolated for further assay. 3 ⁇ L of the lysate supernatant were added to 697 ⁇ L of lysis buffer and the OD 415 determined.
  • the Ca 2+ activated K + channel (Gardos channel) activity was evaluated on whole blood as Rb + influx as described. Whenever indicated the activity of the Gardos channel was evaluated in red cells from BDA-410 treated and untreated mice in the presence of the protein kinase inhibitor calphostin C (1 ⁇ )(Rivera, A. et al., Blood, 99:357-603, 2002; Rivera, A. et al., Am. J. Physiol., 277(4 Pt 1 ):C746-754, 1999; de Franceschi, L. et al., Proteomios, 8:4695-4708, 2008).
  • the K-CI cotransport activity was evaluated in fresh red cells as chloride and volume dependent K + efflux (de Franceschi, L. et al., Am. J. Physiol., 269(4 Pt 1 ):C899-906, 1995).
  • Red cell ghosts were prepared by lysing 1 volume of packed red cells in 10 volumes of ice cold Phosphate Lysis Buffer (in mM PLB: 5 Na 2 HP0 4 pH 8.0, containing a protease inhibitor cocktail tablet, 3 benzamidine, 1 Na 3 V0 4 ) and in presence of NEM (100 mM)( Matte, A. et al., Free Radio. Biol. Med., 49:457-466, 2010; Biondani, A. et al., Proteomios Clin. ⁇ , 2:706-719, 2008). Samples were incubated for 10 min on ice and centrifuged for 10 min at 12,000g, 4C.
  • Phosphate Lysis Buffer in mM PLB: 5 Na 2 HP0 4 pH 8.0, containing a protease inhibitor cocktail tablet, 3 benzamidine, 1 Na 3 V0 4
  • NEM 100 mM
  • a search for calpain-1 cleavage site(s) was carried out with the tool provided by the CaMPDB (DuVerle, D. et al., PLoS One, 6(5):e19035, 201 1 ).
  • a high score was set to avoid false positive results as much as possible.
  • BDA-410 In vivo calpain-1 inhibition by BDA-410 ameliorates red cell pathology in sickle cell SAD mice. Findings addressed whether oral administration of the calpain-1 inhibitor BDA-410 affects the hematological phenotype of sickle cell disease. BDA-410 was administered to the SAD mouse model for SCD that exhibits red cell dehydration. The main advantages of SAD mice for this purpose are: (i) the absence of thalassemic features; (ii) previous successful use to study the mechanisms involved in generation of dense dehydrated red cells; and (iii) lack of the intense hemolysis that obscures red cell dehydration in more severe sickle cell mouse models.
  • mice underwent 14 days treatment with the calpain-1 specific inhibitor BDA- 410 at the dosage of 30 mg/Kg/day by gavage. Hematological parameters were evaluated at baseline and at 7 and 14 days of treatment. No changes in body weight were observed during the treatment period. As shown in Table II, BDA-410 induced a significant increase in hematocrit (Hct) at 7 and 14 days of treatment in SAD mice but not in wild-type. There were also no changes in vehicle-treated mice. No changes in hemoglobin (Hb) levels were present in BDA-410 treated wild-type and SAD mice compared to either baseline values. The mean corpuscular volume (MCV) values significantly increased in both mouse strains at 7 and 14 days of treatment (Table II).
  • MCV mean corpuscular volume
  • MCHC mean corpuscular hemoglobin concentration
  • BDA-410 administration had a time dependent beneficial effect on red cell morphology in SAD mice compared to before treatment.
  • Red cell K + content was significantly reduced in untreated SAD mice compared to wild- type mice, with no major difference in red cell Na + content (FIG. 1 C).
  • the red cell K + content in SAD mouse red cells increased significantly compared to baseline values, while no changes were evident in wild-type mice (FIG. 1 C). No significant changes in red cell Na + content were evident (FIG. 1 C).
  • BDA-410 treatment reduces the activity of the Ca 2+ activated K + channel. Since human dense sickle red cells showed evidence of increased calpain-1 activity and since SAD mice treated with BDA-410 exhibit amelioration of red cell indices and dehydration, a bioinformatic approach was used to search for possible calpain-1 targets on membrane ion transport systems involved in regulation of red cell hydration. Calpain-1 appears to recognize the overall 3- dimensional structure of its substrates more than the primary structure. In addition, calpain-1 generally cleaves substrates by cutting their interdomain regions, indicating its modulatory functions for substrate proteins.
  • the activity of the Gardos channel might be regulated by phosphorylation/dephosphorylation events, involving protein kinase C and phosphatases, whose identity is still unknown.
  • Prx2 membrane association in wild-type and SAD mice was evaluated before and after BDA-410 treatment. As shown in the lower panel of FIG. 3C, Prx2 associated to the membrane is higher in SAD red cell membranes compared to wild-type. BDA-410 treatment significantly reduced the amount of Prx2 associated to the membrane in SAD mice, while no differences were observed in treated wild-type mice (FIG. 3C, lower panel, lanes 2, 4).
  • BDA-410 treatment prevents hypoxia-induced red cell dehydration in SAD mice.
  • Untreated SAD mice exhibited hypoxia-induced red cell dehydration (FIG. 4A and Table 1 ) and red cell K + loss (FIG. 4A and Table 1 ), which were ameliorated by BDA-410 treatment (D 20 1 ,1 10 ⁇ 0.0005 in untreated SAD mice vs 1 ,104 ⁇ 0.001 in BDA-410 treated SAD mice; P ⁇ 0.05, n 6). No changes in reticulocyte count were observed in either mouse group after exposure to hypoxia.
  • Calpain-1 is a non-lysosomal calcium dependent protease that modulates multiple cell functions in various cell models through the selective cleavage of proteins. Calpain-1 is tightly regulated by activator and inhibitor proteins (Hanna, R. et al., Nature, 456:409-412, 2008;
  • the activator protein promotes calpain conformational changes favorable for calpain activation, while the inhibitor protein, calpastatin, blocks the calpain active sites by wedging into the active site, and thus preventing the catalytic cysteine of calpain from reaching the substrate.
  • calpastatin activity is associated with reduced calpastatin content in dense sickle red cells, suggesting an
  • activator/inhibitor unbalance in favor of calpain-1 activation in dense sickle red cells.
  • Prx2 and Gardos channel have putative calpain-1 binding sites and their functions are affected by the calpain-1 inhibitor, indicates calpain-1 is likely involved in the Prx2-Gardos channel network.
  • a possible functional connection between Prx2 and Gardos channel could be linked to the role of Prx2 in protecting crucial cysteine residues from oxidation.
  • the Gardos channel immediately after the pore region, presents a motif
  • Prx2 switches on/off the thiol-redox center of Gardos channel, similar to that described for Prx1 in controlling neuronal differentiation by thiol-redox dependent activation of GDE2 (Yan, Y. et a!., Cell, 138:1209-1221 , 2009).
  • Mature erythrocytes express calpain-1 , an isoform that is tightly regulated by calpastatin, the endogenous inhibitor of calpains.
  • a calpain-1 gene deletion results in improved erythrocyte deformability without any measurable effect on erythrocyte lifespan in vivo.
  • the calcium- induced spheroechinocyte shape transition is compromised in the calpain-1 null erythrocytes.
  • the erythrocyte membrane proteins ankyrin, band 3, protein 4.1 , adducin, and dematin were degraded in the calcium-loaded normal erythrocytes but not in the calpain-1 null erythrocytes.
  • the integrity of spectrin and its state of phosphorylation was not affected in the calcium-loaded erythrocytes of either genotype.
  • the activity of major membrane transporters was measured.
  • the activity of K-CI cotransporter and the Gardos channel was significantly reduced in the calpain-1 null erythrocytes as compared to wild type cells.
  • the basal activity of the calcium pump (plasma membrane Ca2+-ATPase or PMCA) was reduced in the absence of calmodulin in calpain-1 null erythrocyte plasma membrane.
  • Antibodies against spectrin, ankyrin, band 3, protein 4.1 , dematin, and p55 were generated.
  • a monoclonal antibody against calpain was purchased from NOVACASTRA, and a monoclonal antibody (5F10) against erythrocyte calcium pump (Ca 2+ -ATPase) was obtained.
  • the calcium ionophore A23187 was purchased from Calbiochem (La Jolla, CA).
  • Radioisotope ( 32 P) was purchased from DuPont New England Nuclear.
  • PMA, DMSO, Bt 2 cAMP, and other common reagents were purchased from Sigma (St. Louis, MO).
  • reticulocytes were counted after staining with new methylene blue (manual count). Blood cell and reticulocyte number were determined using an automated hematological analyzer (ADVIA 120; Multispecies software; Siemens Diagnostic Solutions, Tarrytown, New York, USA).
  • Blood was collected into acid citrate dextrose anticoagulant and used within 24 hours. Approximately 850 ⁇ L blood was drawn from each mouse via inferior vena cava while anesthetized using isofluorane. Blood was transferred to 1 .5 mL microcentrifuge tubes and spun at 1 ,000 g for 5 minutes. The plasma and buffy coat were removed by aspiration. The remaining RBCs were washed with equal volume of PBS and spun again at 1 ,000 g for 5 minutes. The RBCs were then transferred to 15 mL tubes and washed twice using PBS and centrifugation at 1 ,500 g for 5 minutes.
  • the cells were suspended in 1 .0 mL calcium-free Tyrode's buffer (10 mM HEPES, 12 mM NaHC0 3 , pH 7.5, 137 mM NaCl , 2.5 mM KCI, 5 mM glucose, 0.1 % BSA) and counted using a hemocytometer. The samples were normalized to 8x 10 6 cells/mm 3 with Tyrode's buffer. Experimental samples were prepared using 200 ⁇ L of RBC suspension and 800 ⁇ L of Tyrode's buffer with A23187 added to a final concentration of 1 .0 ⁇ , with and without 50 ⁇ calcium. The RBCs were incubated at 37C for 20 minutes, spun down at 1 ,000 g for 5 minutes, and suspended in 100 ⁇ L of Tyrode's buffer.
  • Tyrode's buffer 10 mM HEPES, 12 mM NaHC0 3 , pH 7.5, 137 mM NaCl , 2.5 mM KCI, 5 mM glucose,
  • RBC deformability was assessed using a laser diffraction Ektacytometer with a thin micro-channel as the shearing geometry (RheoScan-D300; RheoMeditech, Seoul,
  • RBC suspension (6 ⁇ L) of each genotype was mixed with the PBS-PVP (polyvinylpyrrolidone) aliquot (600 ⁇ L volume).
  • the PBS-PVP solution was provided as part of the commercial kit with RheoScan-D300.
  • RBC mixture 500 ⁇ L was transferred to the cartridge and tested using RheoScan-D300. Each sample was tested at least 3 times each in this manner.
  • the RheoScan-D300 measures length and width of the RBCs at 16 shear stress levels from 20 to 0.5 Pa using a CCD camera and computer to measure the elongation index (El).
  • the filtration was started at a pressure of 150 mm H 2 O, and the percent of the flow rate (mL/min) of the erythrocyte suspension (0.1 % hematocrit) in HEPES buffer (96 mM KCI, 64 mM NaCl , 10 mM glucose, 1 .0 mM MgCI 2 , and 16 mM HEPES) relative to that of the aqueous suspending medium at 100 mm H 2 0 was taken as the index of erythrocyte filterability.
  • HEPES buffer 96 mM KCI, 64 mM NaCl , 10 mM glucose, 1 .0 mM MgCI 2 , and 16 mM HEPES
  • Osmotic fragility assay was performed on freshly obtained erythrocytes from 4-6 months old wild type and calpain-1 null mice in the presence of heparin as an anticoagulant. Blood was washed three times with PBS, and resuspended in PBS at 20% hematocrit, followed by incubation with either 1 .0 ⁇ of calcium ionophore A23187, or 1 .0 ⁇ A23187 plus 100 ⁇ CaCI 2 for 15 min at 37C.
  • Erythrocytes were washed twice with the ice-cold salt solution (154 mM NaCl, 9.5 mM Na 2 HP0 4 , 1 .5 mM NaH 2 P0 4 , pH 7.4; 100% salt solution) to terminate calcium loading.
  • Erythrocyte lysis was monitored by the release of hemoglobin into the supernatant upon incubation of cells with graded salt concentrations (100-1 %) for 30 minutes at room temperature. Erythrocyte suspensions were centrifuged, and absorbance of the supernatant was recorded at 540 nm. Each sample in water was taken as 100% erythrocyte lysis, and readings of the same sample in various osmolarity solutions were normalized.
  • Erythrocyte shape change assay was performed essentially as described (White, J., Am. J. Pathol., 77:507-18, 1974). Briefly, blood from 6 wild type and 6 calpain-1 null mice was drawn into a syringe containing 3.8% trisodium citrate (1 :10 v/v). Blood pooled from each genotype was centrifuged at 100xg for 10 minutes. The erythrocyte pellet was washed three times and resuspended in Hanks' buffered salt solution (HBSS, pH 7.3) containing 0.1 % BSA but devoid of calcium.
  • HBSS Hanks' buffered salt solution
  • a final spin of 800xg for 10 minutes was performed to pack the erythrocytes, and the cells were resuspended in the previously mentioned media to produce a hematocrit of 1 %.
  • Each sample was incubated at 37C without calcium, and with 50 ⁇ or 1 .0 mM CaCI 2 in the presence of 1 .5 ⁇ calcium ionophore A23187 for up to 60 minutes.
  • Erythrocyte samples were then placed onto a siliconized glass slide, and visualized by the Differential Interference Contrast (DIC) microscopy using a 100 ⁇ objective on a Nikon Eclipse TE2000-E inverted microscope. Ten random fields of acquired images were analyzed for quantification of erythrocyte morphology at different time intervals. The images are
  • Erythrocyte membrane proteins from ghosts were separated on 8% polyacrylamide gels and stained with Coomassie Blue.
  • proteins were transferred to a nitrocellulose membrane (BioRad), blocked with 5% milk in Tris-HCl buffered saline containing 0.1 % Tween-20, and incubated with the respective antibodies.
  • Enhanced chemiluminescence detection was performed using the SuperSignal West Pico kit (Pierce).
  • the erythrocyte suspension was further incubated for 10-20 minutes at 37C, and incubation was terminated by cell lysis in 10 volumes of ice-cold lysis buffer (5 mM sodium phosphate buffer, pH 8.0, 1 .0 mM EDTA and 1 .0 mM PMSF).
  • Erythrocyte ghosts were prepared, resuspended in the SDS sample buffer, and analyzed by SDS-PAGE and Western blotting.
  • Fresh erythrocytes were resuspended at 20% hematocrit in solution A (145 mM NaCl , 5.0 mM KCI, 10 mM HEPES, pH 7.4, 0.1 mM Na 2 HP0 4 ), and incubated at 37C with 0.4 mCi/mL of ( 32 P) orthophosphate for 2.0 hours to label the intracellular ATP pool. Aliquots (250 ⁇ L) of cells were then treated with either 2.0 mM Bt 2 cAMP, or 1 .0 ⁇ PMA, or 1 .0 ⁇ calcium ionophore A23187 plus 500 ⁇ of CaCI 2 at 37C for indicated time intervals.
  • solution A 145 mM NaCl , 5.0 mM KCI, 10 mM HEPES, pH 7.4, 0.1 mM Na 2 HP0 4
  • Aliquots (250 ⁇ L) of cells were then treated with either 2.0 mM Bt 2 cAMP, or 1 .0 ⁇ PMA
  • Erythrocytes were washed once with the ice-cold solution A, and then immediately lysed with 10 volumes of ice- cold lysis buffer as described above. Erythrocyte ghosts were washed twice with the lysis buffer, solubilized in SDS sample buffer, and radiolabeled proteins were visualized by 8% SDS- PAGE and autoradiography.
  • hemoanalyzer (Siemens Diagnostic Solutions, Tarrytown, NY). Plasma and buffy coat were removed after passing through cotton, and cells were centrifuged at 2,500 rpm for 4 minutes at 4C. The red cell pellet was washed four times with an ice-cold choline wash solution (CWS) containing 172 mM choline chloride, 1 .0 mM MgCI 2 , 10 mM TRIS-MOPS, pH 7.4 at 4C as described. An aliquot of cells was then suspended at 50% volume with CWS-Mg 2+ free, and determinations of hematocrit and cell Mg 2+ (1 :50 dilution) were performed.
  • CWS ice-cold choline wash solution
  • KCC1 K-CI cotransporter
  • the activity of K-CI cotransporter (KCC1 ) was calculated as the difference between K + efflux in hypotonic NaCl (100 mM), isotonic NaCl (160 mM), and hypotonic sodium sulfamate (100 mM) media (Bize, I. et al., Am. J. Physiol. Cell Physiol., 285:C31 -38, 2003). Fluxes were measured between 5 and 35 minutes at 37C, and the flux was estimated from the slope of the time course curve. The maximal rates of K-CI cotransporter activity was calculated from the difference between the volume-sensitive fraction and the Cl-dependent K + flux, and expressed as mmol/10 3 cells ⁇ hour.
  • Gardos channel activity Freshly washed erythrocytes were suspended at 2% hematocrit in isotonic influx media containing 165 mM NaCl, 2.0 mM KCI, 0.15 mM MgCI 2 , 1 .0 mM ouabain, 10 mM Tris-MOPS, pH 7.4 at 22C, 10 ⁇ bumetanide, and 10 ⁇ Ci/mL 86 Rb in the presence or absence of 50 nM charybdotoxin, a specific blocker of the Gardos channel. Free Ca 2+ in the influx media was buffered to 7.0 ⁇ with 1 .0 mM citrate buffer as described before.
  • radioactivity was measured in a gamma counter (model 41600 HE; Isomedic ICN-MP
  • mice erythrocyte membranes were isolated from freshly collected blood (Zaidi, A. et ai, Biochim. Biophys. Acta., 1236:1 14-8, 1995). Briefly, whole blood was centrifuged at 3,000 rpm for 15 minutes. Plasma and buffy coat were carefully aspirated off and erythrocytes were lysed in the hypotonic buffer containing 10 mM Tris-HCl , pH 6.7 and 0.5 mM EDTA. Membranes (ghosts) were centrifuged several times at 10,000 rpm for 20 minutes in a JA20 rotor to remove hemoglobin.
  • Ghosts were then washed using a hypertonic buffer containing 10 mM HEPES, pH 7.2, 120 mM KCI, 0.5 mM MgCI 2 , 50 ⁇ CaCI 2 and centrifuged until free of hemoglobin. Protein concentration was determined using a BCA kit (Pierce). Ghosts were divided into small aliquots and frozen at -80C until further analysis.
  • PMCA activity Erythrocyte membranes isolated from the wild type and calpain-1 null mice were assayed for PMCA activity in a 96-well plate using the Malachite Green colorimetric assay (Lanzetta, P. et al., Anal. Biochem., 100:95-97, 1979; Zaidi, A. et al., Free Radic. Biol. Med., 47:1507-14, 2009).
  • Activity of PMCA was determined by measuring the release of inorganic phosphate by calcium-dependent ATP hydrolysis.
  • Each well in a final volume of 100 ⁇ L, contained 25 mM Tris-HCl , pH 7.4, 50 mM KCI, 1 .0 mM MgCI 2 , 0.1 mM ouabain, 4.0 ⁇ g/mL oligomycin, 200 ⁇ EGTA and CaCI 2 to give the desired final free Ca 2+ concentration (ranging from 1 -10 ⁇ ).
  • the final free Ca 2+ concentration was calculated using the software available at calcium.com, which calculates multiple equilibria between all ligands in solution.
  • the PMCA activity measured in the presence of calcium without calmodulin (CaM) is referred to as the 'basal' activity, and that measured in the presence of calcium and CaM (340 nM) as "CaM-stimulated' activity.
  • the reaction was started by the addition of ATP (1 .0 mM final concentration), continued for 30 minutes at 37C, and stopped by the addition of Malachite Green dye.
  • the contents were made acidic by the addition of 19.5% H 2 S0 4 , incubated for 45 minutes, and color measured at 650 nm in a plate reader.
  • the PMCA activity was defined as the calcium-activated ATP hydrolysis and expressed as nanomoles of inorganic phosphate (Pi) liberated per mg protein per minute based on the values from a standard curve of the absorbance at various concentrations of free inorganic phosphate.
  • Calpain-1 null mice were generated by targeted mutagenesis of the Capnl gene.
  • Targeted deletion of eight amino acids was introduced within exon 4 of the Capnl gene, followed by the insertion of the gene disrupting pGK-Neo cassette.
  • the calpain-1 null mice were backcrossed more than 20 generations on pure C57BL/6 genetic background. Mature erythrocytes isolated from adult mice were analyzed for calpain activity. Erythrocytes, free of platelets and leukocytes, were assayed for total calpain activity by casein zymography.
  • calpain-1 null erythrocytes were deficient of any detectable protease activity by casein zymography (data not shown), consistent with findings using PCR and casein zymography assays.
  • purified erythrocyte membranes (ghosts) free of hemoglobin were also completely deficient of any calpain activity by casein zymography. Since both erythrocyte calpains migrate at the same position in the SDS-polyacrylamide gels under reducing
  • erythrocytes were purposely loaded with high concentrations of calcium for two reasons: (a) unlike human erythrocytes, the mouse erythrocyte membrane proteins were relatively resistant to calcium-induced degradation under these conditions; (b) it was useful to examine if other calcium-dependent proteases exist that might be activated at high local calcium concentrations, thus compensating for calpain-1 loss in the KO erythrocytes.
  • Erythrocyte membrane proteins including ⁇ -spectrin, are phosphorylated by multiple protein kinases in intact erythrocytes.
  • ⁇ -spectrin phosphorylated by multiple protein kinases in intact erythrocytes.
  • intact erythrocytes were metabolically labeled with radiolabeled phosphate.
  • Protein kinases were activated with dibutyryl cAMP, an activator of cAMP-dependent protein kinase (PKA), phorbol myristic acid (PMA), an activator of protein kinase C (PKC), and calcium (500 ⁇ ) plus calcium ionophore A23187 (1 .0 ⁇ ) to activate calcium-dependent kinases as well as calpain-1 .
  • PKA cAMP-dependent protein kinase
  • PMA phorbol myristic acid
  • PLC protein kinase C
  • calcium (500 ⁇ ) plus calcium ionophore A23187 (1 .0 ⁇ ) to activate calcium-dependent kinases as well as calpain-1 .
  • Radiolabeled erythrocytes were incubated for 30 minutes at 37C, and phosphorylation of membrane proteins was examined by SDS-PAGE followed by autoradiography (FIG. 5C). The calpain-1 loss did not cause any change in the
  • Calpain-1 null erythrocytes loaded with 50 ⁇ calcium were significantly more susceptible to echinocyte and spheroechinocyte shape transition as compared to the WT erythrocytes over the 60-minute incubation period (FIG.
  • Calpain-1 suppresses erythrocyte deformability and filterability
  • calpain-1 degrades key proteins of the erythrocyte membrane cytoskeleton
  • WT and KO erythrocytes were incubated with calcium ionophore A23187 (1 .0 ⁇ ) in calcium-free Tyrode's buffer (10 mM HEPES, 12 mM NaHC0 3 , pH 7.5, 137 mM NaCl, 2.5 mM KCI, 5 mM glucose, 0.1 % BSA), and loaded with 50 ⁇ calcium for 10 minutes at 37C.
  • Erythrocytes were then subjected to increasing shear stress, and their deformability was measured in the Rheoscan- D300 Ektacytometer (FIG. 7A).
  • the maximum value of the deformability index (Dl max) a rough measure of the erythrocyte membrane deformability at the optimal surface to volume ratio, progressively increased with the shear stress (FIG. 7A).
  • the deformability index was significantly higher in the calpain-1 null erythrocytes as compared to WT erythrocytes (FIG. 7A).
  • the calcium loading caused a precipitous decline in the erythrocyte deformability of both genotypes (FIG. 7A).
  • the deformability index was significantly higher in the calpain-1 null erythrocytes as compared to the WT mice (FIG. 7A).
  • the filterability of the erythrocytes was measured through a 4.6-micron nickel mesh (FIG. 7B).
  • the filtration rate of WT and calpain-1 null erythrocytes was similar when both cell types were analyzed in the absence of calcium loading (FIG. 7B).
  • the filtration rate of calpain-1 null erythrocytes was reproducibly higher as compared to WT erythrocytes (FIG. 7B).
  • the mean filterability of the calpain-1 null erythrocytes was 18% higher than control erythrocytes, indicating that the absence of calpain-1 protects the cells against a calcium-induced decline in filtration rate.
  • the filterability of both types of erythrocytes declined to zero, suggesting that the dominant calcium-dependent factor controlling erythrocyte filterability is still retained in the calpain-1 null erythrocytes.
  • FIG. 8A, B The effect of calcium influx on osmotic fragility of erythrocytes was assessed at different salt concentrations. Washed erythrocytes treated with 1 .0 ⁇ calcium ionophore A23187 were loaded with 100 ⁇ calcium for 15 minutes at 37C, and cell lysis was measured by hemoglobin release. Although the influx of calcium into erythrocytes did shift the fragility curve in both genotypes, the statistical difference between the hemolysis of WT and KO erythrocytes was not significant (FIG. 8A). However, in several osmotic fragility measurements, it was noticed that the extent of hemolysis was slightly higher in the calpain-1 null erythrocytes at a particular salt concentration.
  • FIG. 8B the hemolysis profile of freshly isolated erythrocytes was compared with no exposure to ionophore and calcium (FIG. 8B). This step was taken to potentially eliminate the detrimental effects of incubation period on erythrocyte properties. Overall, the hemolysis pattern was similar between the WT and KO erythrocytes at various salt concentrations (FIG. 8B). A small increase in the hemolysis of calpain-1 null erythrocytes at 200 millimolar salt concentration as compared to WT erythrocytes was consistently observed, however (FIG. 8B).
  • K-CI cotransport is deregulated in calpain-1 null erythrocytes
  • Calpain-1 differentially regulates erythrocyte calcium pump.
  • PMCA activity was measured in erythrocyte membranes (ghosts) over a range of free calcium concentrations (0-10 ⁇ ).
  • the calcium pump activity was measured in the presence of inhibitors of other known ATPases, thus ensuring the specificity of PMCA activity.
  • the activity of the calcium pump was measured both in the absence and presence of exogenous calmodulin, a physiological regulator of the calcium pump (Jarrett, H. & and Penniston, J., Biochem. Biophys. Res. Commun., 77:1210-6, 1977).
  • the results indicate a linear increase in the pump activity with increasing calcium concentrations followed by a plateau (FIG.
  • V max values in the wild type and calpain-1 null ghosts were 8.56 ⁇ 0.31 and 4.07 ⁇ 1 .3, in the absence of calmodulin, and 20 ⁇ 2.02 versus 22.9 ⁇ 0.9, in the presence of calmodulin, respectively (FIG. 9C).
  • the difference in V max values between wild type and KO in the absence of calmodulin were statistically significant as determined by the Mann-Whitney Rank Sum Test (p ⁇ 0.029).
  • Atomic force microscopy reveals a protective role of calpain-1 inhibition on membrane cytoskeleton.
  • AFM topographic images of wild type erythrocyte membrane revealed an intact cytoskeleton network with fine filament structure (FIG. 10, top panels) similar to other reports (Liu, F. et al., Cell Biochem. Biophys., 38:251 -70, 2003; Liu, F. et al., Scanning, 33:426- 36, 201 1 ).
  • Influx of 50 ⁇ calcium in erythrocytes via calcium ionophore A23187 caused damage of the cytoskeleton integrity as evident by the absence of fine structure and elevated cytoskeleton height and grain size (FIG. 10, Table 2).
  • AFM images of calcium-treated calpain-1 null erythrocytes also showed loss of fine structure but the changes in the cytoskeleton height and grain size were much less pronounced than those observed in the calcium-treated wild type controls (FIG. 10, Table 2). Consistent with the erythrocyte deformability data (FIG. 7A), the loss of calpain-1 provided protection from the calcium-induced cytoskeleton instability as 50 ⁇ calcium did not produce any significant change in the cytoskeleton height and grain size (Table 2). These results suggest that the loss of calpain-1 only slightly modifies the cytoskeletal network under steady state conditions, and offers significant protection under conditions of calcium-induced activation of calpain-1 activity in intact erythrocytes.
  • calpain activity traditionally refers to the cumulative activity of two calpains- Calpain-1 , also known as ⁇ -calpain, which is activated at micromolar calcium concentration, and calpain-2, designated as m-calpain, which is activated at millimolar calcium concentration in vitro.
  • calpain polypeptides with overlapping substrate specificity are differentially expressed in mammalian cells.
  • An exception to this rule is the adult erythrocyte where the total calpain activity is contributed exclusively by calpain-1 .
  • Calpain-1 causes limited cleavage of PKC to generate PKM (Kishimoto, A. et al., J. Biol.
  • the PKC serves as a substrate of calpain-1 since its degradation is blocked in calpain-1 null erythrocytes activated by either calcium or PMA (FIG. 5C).
  • phosphorylation of ⁇ -spectrin and other substrates remains unaltered despite partial degradation of PKC in normal erythrocytes (FIG. 5C).
  • calpain-1 A functional role of calpain-1 has been indicated in the regulation of erythrocyte shape change.
  • Treatment of erythrocytes with a calpain inhibitor blocked their transition from echinocytes to spherocytes.
  • purified cytoplasmic domain of band 3 is known to inhibit erythrocyte membrane shape change in vitro (Carter, D. & Fairbanks, G., J. Biol. Chem., 24:385-93, 1984).
  • Results described herein are consistent with the overall conclusion that calpain-1 functions as a key regulator of erythrocyte shape change (FIG. 6).
  • the findings presented herein unveil some new aspects of erythrocyte shape regulation by calpain- 1 .
  • Calpain-1 loss not only modulates the final erythrocyte shape change but also affects the rate of shape transition in a calcium-dependent manner (FIG. 6).
  • the discocyte to echinocyte shape transition is potentiated at lower calcium concentration (FIG. 6A). At higher calcium concentration, the rate of discocyte-echinocyte transition is further
  • erythrocytes The shape and mechanical properties of erythrocytes are thought to be partially controlled by the submembrane network referred to as the membrane skeleton (Bennett, V. & Gilligan, D., Ann. Rev. Cell Biol., 9:27-66, 1993). Regulatory modifications of skeletal proteins such as oxidation, proteolysis and phosphorylation influence the mechanical properties by altering critical protein interactions. Because calcium-induced phosphorylation of membrane proteins remains normal in calpain-1 null erythrocytes (FIG. 5C), the KO model system allows a direct evaluation of the impact of moderate skeletal protein proteolysis on membrane
  • hematological parameters such as bilirubin, iron, heme and LDH are unaffected in the KO mice.
  • the reticulocyte count was also normal in calpain-1 null mice.
  • erythrocyte mean cell volume (MCV) was measured in six adult mice of each genotype. A small but statistically significant increase in the MCV of KO mice was found (Table 3).
  • calpain-1 loss may trigger down-regulation of erythrocyte membrane-associated protein phosphatases thus causing inhibition of the K-CI cotransporter (Bize, I. et ai, J. Membr. Biol., 177:159-68, 2000; Bize, I. et ai., Am. J. Physiol., 277:C926-36, 1999).
  • PMCA integral membrane protein referred to as the PMCA (Carafoli, E., J. Biol. Chem., 267:21 15-8, 1992; Brini, M. & Carafoli, E., Physiol Rev., 89:1341 -78, 2009).
  • the PMCA utilizes metabolic energy from ATP hydrolysis to transport Ca 2+ across the plasma membrane against a 10,000-fold gradient, thus playing a critical role in maintaining precise levels of intracellular calcium.
  • the PMCA is a -140 kDa protein with 10 transmembrane helices and several intracellular domains including its active site located between transmembrane segments 4 and 5.
  • the C-terminal end of the PMCA contains an autoinhibitory domain, which interacts with its catalytic site preventing the binding and utilization of ATP thus keeping the enzyme in a state of low activity (Falchetto, R. et al., J. Biol. Chem., 266:2930-6, 1991 ).
  • calmodulin a calcium-binding protein
  • binding of calmodulin (CaM) to PMCA triggers the dissociation of the autoinhibitory domain away from the catalytic core and release of autoinhibition, thus stimulating PMCA activity several-fold and providing the driving force for Ca 2+ transport across the plasma membrane
  • calmodulin a calcium-binding protein
  • FIG. 9 differential regulation of PMCA activity and calmodulin content in calpain-1 null erythrocytes may offer a potential mechanistic explanation for the regulation of erythrocyte cell shape, deformability, filterability, and mean cellular volume.

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Abstract

La présente invention concerne des compositions et des méthodes de traitement de la drépanocytose, ainsi que des procédés d'identification d'agents qui permettent de traiter ou de prévenir la drépanocytose.
PCT/US2012/063404 2011-11-02 2012-11-02 Identification et utilisation d'inhibiteurs de protéase pour traiter ou prévenir une drépanocytose WO2013067415A1 (fr)

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