EP4633622A2 - Compositions and methods for regulating procoagulant activity and thrombosis - Google Patents

Compositions and methods for regulating procoagulant activity and thrombosis

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Publication number
EP4633622A2
EP4633622A2 EP23904458.9A EP23904458A EP4633622A2 EP 4633622 A2 EP4633622 A2 EP 4633622A2 EP 23904458 A EP23904458 A EP 23904458A EP 4633622 A2 EP4633622 A2 EP 4633622A2
Authority
EP
European Patent Office
Prior art keywords
day
tmem16
inhibitors
benzbromarone
therapeutically effective
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
EP23904458.9A
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German (de)
French (fr)
Inventor
Alec A. SCHMAIER
Sol SCHULMAN
Thomas Christian Lines
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Quercis Pharma AG
Beth Israel Deaconess Medical Center Inc
Original Assignee
Quercis Pharma AG
Beth Israel Deaconess Medical Center Inc
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Publication of EP4633622A2 publication Critical patent/EP4633622A2/en
Pending legal-status Critical Current

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/02—Antithrombotic agents; Anticoagulants; Platelet aggregation inhibitors
    • 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
    • A61K31/045—Hydroxy compounds, e.g. alcohols; Salts thereof, e.g. alcoholates
    • A61K31/05—Phenols
    • A61K31/055—Phenols the aromatic ring being substituted by halogen
    • 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
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/34—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having five-membered rings with one oxygen as the only ring hetero atom, e.g. isosorbide
    • A61K31/343—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having five-membered rings with one oxygen as the only ring hetero atom, e.g. isosorbide condensed with a carbocyclic ring, e.g. coumaran, bufuralol, befunolol, clobenfurol, amiodarone
    • 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
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/35—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom
    • A61K31/352—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom condensed with carbocyclic rings, e.g. methantheline 
    • A61K31/353—3,4-Dihydrobenzopyrans, e.g. chroman, catechin
    • 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
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
    • A61K31/403—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil condensed with carbocyclic rings, e.g. carbazole
    • A61K31/404—Indoles, e.g. pindolol
    • 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
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/425—Thiazoles
    • A61K31/426—1,3-Thiazoles
    • 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
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/54—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one sulfur as the ring hetero atoms, e.g. sulthiame
    • A61K31/5415—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one sulfur as the ring hetero atoms, e.g. sulthiame ortho- or peri-condensed with carbocyclic ring systems, e.g. phenothiazine, chlorpromazine, piroxicam
    • 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
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
    • A61K31/7048—Compounds having saccharide radicals and heterocyclic rings having oxygen as a ring hetero atom, e.g. leucoglucosan, hesperidin, erythromycin, nystatin, digitoxin or digoxin

Definitions

  • the present disclosure relates to treatment of thrombosis and related disorders using
  • TMEM16 inhibitors alone or in combination.
  • the present invention relates to treatment of thrombosis.
  • TMEM16 proteins contribute to procoagulant activity in platelets, epithelial cells, and endothelial cells.
  • Two phospholipid scrambiases are identified that contribute to processes that lead to thrombosis, including TMEM16F and TMEM16E.
  • TMEM16E is newly identified in this invention as a contributor to thrombotic processes, and is involved in fibrin generation, the terminal product in coagulation activation.
  • Targeting TMEM16 proteins with compounds that inhibit their activity reduces thrombosis without increasing bleeding complications. This invention highlights importance of modulation of TMEM16 inhibition for prevention and treatment of thrombotic diseases.
  • TMEM16 proteins are expressed in cells involved in physiological pathways that can lead to thrombosis, including both epithelial and endothelial cells.
  • Endothelial cells ECs
  • ECs Endothelial cells
  • coagulation proteins require a membrane surface containing anionic phospholipid, most notably phosphatidylserine (PS).
  • PS can be rapidly externalized to the outer cell membrane leaflet by phospholipid “scrambiases”, such as TMEM16F. How ECs externalize phospholipids to support coagulation is not understood.
  • TMEM16 phospholipid scrambiases may be a valuable therapeutic target for thrombotic cardiovascular disease.
  • Thrombotic disorders such as myocardial infarction, stroke, and venous thromboembolism are leading causes of mortality worldwide (Wendelboe and Raskob, Circ Res 118: 1340-7 (2016)). Blood coagulation, and therefore thrombosis, depends at key stages on a membrane surface containing anionic phospholipid, most commonly phosphatidyl serine (PS) (Zwaal et al., Acta - Rev Biomembr 1376:433-453 (1998)).
  • PS phosphatidyl serine
  • PS promotes the recruitment and activation of factor X by the tissue factor (TF)-factor Vila complex (Bach and Rifkin, Proc Natl Acad Set USA 87:6995-6999 (1990); Nemerson, J Clin Invest 47:72-80 (1968); Krishnaswamy et al., J Biol Chem 267:26110-26120 (1992)) and assembly and activity of the factor Xa-Va- prothrombin coagulation enzyme complex (Krishnaswamy et al., J Biol Chem 263:3823-3834 (1988); Rosing et al., J Biol Chem 255:274-283 (1980)), the initiating and ultimate steps in thrombin formation, respectively.
  • tissue factor (TF)-factor Vila complex tissue factor (TF)-factor Vila complex
  • Coagulation factor interaction with PS accelerates enzyme kinetics by at least 3 orders of magnitude to physiologic rates (Rosing et al., J Biol Chem 255:274- 283 (1980); Miletich et al., Proc Natl Acad Sci USA 74:4033-4036 (1977)).
  • PS constitutes approximately 10-15% of plasma membrane phospholipid (Leventis and Grinstein, Amu Rev Biophys 39:407-427 (2010)), but under basal conditions is sequestered on the inner membrane leaflet and therefore inaccessible to the extracellular environment.
  • a sustained rise in intracellular calcium (Ca 2+ ) triggers PS extemalization to the outside of the cell (Balasubramanian et al., J Biol Chem 282: 18357-18364 (2007); Bevers et al., Biochim Biophys Acta 736:57-66 (1983)) by Ca 2+ - activated phospholipid scrambiases (PLSs), transmembrane channels that allow PS to move down its concentration gradient to the outer plasma membrane leaflet (Bevers and Williamson, Physiol Rev 96:605-645 (2016)).
  • PLSs Ca 2+ - activated phospholipid scrambiases
  • PS-binding proteins like annexin V and lactadherin prevent enzyme complex assembly and inhibit coagulation in vitro (Andree et al., J Biol Chem 267: 17907-17912 (1992); Ravanat et al., Biochem J 282:7-13 (1992); Shi et al., J. Thromb Haemost 6: 1167-1174 (2008)), and decrease thrombosis in vivo (Shi et al., J. Thromb Haemost 6: 1167-1174 (2008); Thiagarajan and Benedict, Circulation 96:2339-2347 (1997); Rbmisch et al., Thromb Res 61 :93- 104 (1991)).
  • PS exposure is an integral step in thrombosis and that targeting PS may be a viable antithrombotic strategy.
  • activated platelets Following agonist stimulation in vitro, activated platelets readily externalize PS and support thrombin generation (Miletich et al., Proc Natl Acad Sci USA 74:4033-4036 (1977); Bevers et al., Biochim Biophys Acta 736:57-66 (1983)).
  • the transition of platelets into a procoagulant form has provided an accessible means of studying cell-based PS exposure in coagulation. It has therefore been suggested that activated platelets are the major source of procoagulant PS in vivo and thus the major site of coagulation enzyme complex assembly for hemostasis and thrombosis (Heemskerk et al., Thromb Haemost 88: 186-193 (2002)).
  • TMEM16F as a PLS required for PS extemalization in platelets (Suzuki et al., Nature 468:834-840 (2010); Yang et al., Cell 151 : 111-122 (2012)) and the finding that mutations in TMEM16F underlie the mild-to-moderate bleeding disorder Scott syndrome (Suzuki et al., Nature 468:834-840 (2010); Castoldi et al., Blood 117:4399-400 (2011)) launched investigation of TMEM16F in platelet function.
  • Platelets require TMEM16F to externalize PS in response to stimuli that raise intracellular Ca 2+ (Fujii et al., Proc Natl Acad Sci USA 112:12800-12805 (2015)). Loss of phospholipid scrambling in platelets protects from thrombosis or impairs hemostasis in some studies but not in others (Fujii et al., Proc Natl Acad Sci USA 1 12: 12800-12805 (2015); Baig et al., Arterioscler Thromb Vase Biol 36:2152-2157 (2016); Zhao et al., Nat Commun 8: 1-11 (2017); Mattheij et al., FASEB J 30:727-737 (2016)). It is therefore likely that sources of PS beyond the platelet and additional proteins beyond TMEM16F regulate procoagulant phospholipid extemalization.
  • Endothelial cells form a constitutive anticoagulant surface under basal conditions to maintain blood flow. Loss of this anticoagulant property is considered a hallmark of cardiovascular disease leading to thrombosis. However, the relative contribution of activated endothelium to blood clotting in vivo is not well understood.
  • Inflammatory stimuli such as hypoxia, cytokines, and lipopolysaccharide induce PS extemalization on ECs (Ran et al., Cancer Res 62:6132-40 (2002); Gao et al., PLoS One 2015;10: 1-16 (2015); Higgins et al., J Clin Invest 128: 1471-1484 (2016)), but the physiologic significance of EC phospholipid scrambling and the proteins that regulate it have not been characterized. Inflammatory stimuli also induce TF expression in ECs.
  • TF binds factor Vll/VIIa but exists in a deactivated or “cryptic” state (REF), and PS extemalization is at least partly responsible for enhancing the factor Vila cofactor activity of TF (Ruf et al., J Biol Chem 1991;266:2158-2166 (1991); Wolberg et al., Blood Coagitl Fibrinolysis 10:201-210 (1999)). Therefore, identification of regulators of PS extemalization will aid our understanding of TF activation and inflammatory thrombosis.
  • REF deactivated or “cryptic” state
  • a targeted screen was performed of genes encoding proteins predicted to regulate transmembrane phospholipid transport for their contribution to TF-dependent factor Xa generation in ECs.
  • This approach identified two TMEM16 family members, TMEM16F, and its closest paralog, TMEM16E (Whitlock and Hartzell, Annu Rev Physiol 79: 119-143 (2017)), that support EC procoagulant activity.
  • the TMEM16 family are all transmembrane proteins, activated by Ca 2+ , and function as ion channels, PLSs, or in some cases both (Whitlock and Hartzell, Anna Rev Physiol 79:119-143 (2017)).
  • TMEM16E is highly expressed in skeletal muscle, where it regulates muscle regeneration and repair (Whitlock et al., J Gen Physiol 2018;150: 1498-1509 (2016); Griffin et al., Hum Mol Genet 25: 1900-1911 (2016)), but is not expressed in platelets (Fujii et al., Proc Natl Acad Sci USA 112:12800-12805 (2015); Rowley et al., Blood 118:el01-el 11 (2011)) and has no previously known role in hemostasis or thrombosis.
  • Our studies demonstrate that both TMEM16E and TMEM16F controlled PS extemalization in ECs and are necessary to support coagulation on the endothelial surface.
  • FIG. 1A-G shows that TMEM16E and TMEM16F are required for TF activity and thrombin generation on ECs.
  • FIG.1A shows a heat map illustrating the relative positive or negative regulation of factor Vila catalyzed factor X activation following silencing of the indicated genes in HUVECs. Pools of four distinct siRNA were directed at the indicated target and tested in triplicate. Each box represents an independent experimental plate with scale bar depicting percentage of factor Xa generated compared to cells transfected with untargeted control siRNA. Lighter color indicates lower percentage factor Xa generation compared to control.
  • Figs. 1B-G shows that TMEM16E and TMEM16F are required for TF activity and thrombin generation on ECs.
  • FIG.1A shows a heat map illustrating the relative positive or negative regulation of factor Vila catalyzed factor X activation following silencing of the indicated genes in HUVECs. Pools of four distinct siRNA were directed at the indicated target and
  • HUVECs were transfected with individual siRNAs for 72 h and assayed for their ability to support factor Vila catalyzed factor Xa generation (Figs. 1B-D) or thrombin generation in plasma-treated ECs (Figs. 1E-G).
  • Cells were stimulated with TNF-a (10 ng/mL) for 3.5 h (FIG. IB, FIG. 1C, FIG. IE, and FIG. IF), TNF-a for 3.5 h plus Ca 2+ ionophore A23187 (6 pM) for 20 min (FIG. ID) or A23187 alone for 20 min (FIG. 1G).
  • Representative experiments are depicted as mean absorbance for factor Xa generation (FIG.
  • FIG. 2A-F shows that TMEM16E and TMEM16F are required for phosphatidyl serine (PS) external! zati on on ECs.
  • HUVECs were transfected with indicated siRNAs for 72 h, stimulated with TNF-a (10 ng/mL) for 18 h (FIG. 2A) or Ca 2+ ionophore A23187 (6 pM) for 20 min (FIG. 2B), and stained with annexin V (green) to detect PS externalization and Zombie Red (red) to detect cell death.
  • Total annexin V fluorescence for each image was normalized to number of nuclei (blue) and dead (Zombie Red-positive, red) cells.
  • FIG. 2C PS externalization following treatment with ionophore A23187 (6 pM) was detected using annexin V by flow cytometry. Histograms are generated from gating on live (DAPI-negative) cells only.
  • FIG. 2D HUVECs were transfected with indicated siRNAs for 72h, stimulated with TNF-a (10 ng/mL) and stained for TF (green). Mean fluorescent intensity (MFI) was normalized to background for each image. Representative images are shown for each experiment.
  • FIG. 2E and FIG. 2F Mean fluorescent intensity
  • HUVECs were transfected with indicated siRNAs for 72 h, and intracellular Ca 2+ flux was measured with the Ca 2+ -sensitive dye Calbryte 520 AM following stimulation with thrombin (1 U/mL).
  • Silencing of the store-operated Ca 21 regulator STIM1 served as a positive control.
  • 16E, 16F, and TF denote siRNA targeting TMEM16E, TMEM16F, and tissue factor, respectively.
  • #1 and #2 denote different siRNA sequences. Scale bar is 50 pm in FIG.
  • FIG. 3A-G shows that PS externalization visualized via intravital microscopy occurs on the vessel wall and is unaffected by platelet inhibition. Thrombus formation was monitored for 180 seconds in wild-type mice following laser injury of the cremasteric arteriole in the presence or absence of the platelet aggregation inhibitor eptifibatide (10 pg/g body weight).
  • FIG. 3A Representative images at indicated time points of the PS probe annexin V (red, Alexa fluor-647), platelets (anti-CD42b antibody, blue, Dylight 405), and fibrin (anti-fibrin antibody, green, Dylight 488). Note annexin V positivity on the vessel wall and in the absence of platelet aggregation.
  • platelets are labeled blue. Representative images are shown from 10 individual thrombi. Arrowheads denote extent of vessel-wall injury and “X” indicates site of laser ablation. Arrows indicate extension of PS extemalization to the opposite vessel wall. Scale bar is 25 pm.
  • FIG. 4A-L shows that TMEM16E supports vessel wall fibrin formation. Thrombus formation following laser injury of the cremasteric arteriole was monitored for 180 sec in TMEM16E /_ ( ⁇ 4no5' /_ ) or TMEM16E +/+ (Ano5 ) littermate controls in the presence of vehicle (FIG. 4A-D) or eptifibatide 10 pg/g body weight (FIG. 4E-H). Platelet and fibrin accumulation were monitored by anti-CD42b and anti-fibrin antibody conjugated to Dylight 405 and 488, respectively. Kinetics and magnitude of median integrated relative fluorescent units (RFU) for platelet (FIG. 4A and FIG.
  • REU median integrated relative fluorescent units
  • FIG. 4E and fibrin (FIG. 4C and FIG. 4G) accumulation are shown following laser injury.
  • Area under the curve (AUC) for fluorescent intensity was determined for platelets (FIG. 4B and FIG. 4F), and fibrin (FIG. 4D and FIG. 4H) for each thrombus.
  • FIG. 5A-F shows thatTMEM16 antagonists reduce EC procoagulant activity by inhibiting PS extemalization.
  • FIG. 5A HUVECs were treated with Ca 2+ ionophore A23187 at indicated concentrations for 20 min and PS extemalization was determined by annexin V binding, measured by flow cytometry. Histograms are generated from gating on live (D API-negative) cells only.
  • FIG. 5B HUVECs were treated with both TNF-a (10 ng/mL) for 3.5 h and A23187 for 20 min at indicated concentrations and analyzed for factor Vila catalyzed conversion of factor X to factor Xa.
  • FIG. 5C HUVECs were treated with A23187 (6 pM) in the presence of TMEM16 inhibitors CaCCinh-AOl (A01) and benzbromarone (BBR) (10 pM) and analyzed for PS extemalization as in FIG. 5A.
  • FIG. 5D TMEM16 antagonists were assayed for their ability to inhibit factor Xa generation on HUVECs stimulated with TNF-a (10 ng/mL, 3.5 h) followed by A23187 (6 pM, 20 min).
  • FIG. 5E and FIG. 5F FIG. 5E and FIG. 5F.
  • HUVECs were treated with TMEM16 inhibitors A01 or BBR, or the phospholipase C inhibitor U73122, and intracellular Ca 2 transients were measured with Calbryte 520 AM following stimulation with thrombin (1 U/mL).
  • ANO VA with Tukey’s posttest *p ⁇ 0.05, **p ⁇ 0.01, ***p ⁇ 0.001, ****p ⁇ 0.0001.
  • FIG. 6A-L shows that benzbromarone inhibits thrombosis without increasing bleeding.
  • Thrombus formation following laser injury of the cremasteric arteriole was monitored for 180 sec in mice treated with benzbromarone (BBR 5 pg/g) or vehicle (FIG. 6A-D and FIG. 61) and BBR or vehicle in the presence of eptifibatide (10 pg/g body weight) (FIG. 6E-H and FIG. 6J).
  • Platelet and fibrin accumulation were monitored by anti-CD42b and anti-fibrin antibody conjugated to Dylight 405 and 488, respectively.
  • Kinetics and magnitude of median integrated relative fluorescent units (RFU) for platelet FIG. 6A and FIG.
  • FIG. 6E and fibrin (FIG. 6C and FIG. 6G) accumulation are shown following laser injury.
  • Area under the curve (AUC) for fluorescent intensity was determined for platelets (FIG. 6B and FIG. 6F), fibrin (FIG. 6D and FIG. 6H) for each thrombus.
  • FIG. 61 and FIG. 6J Injury sizes associated with the thrombi analyzed above, analyzed by Student’s t test.
  • FIG. 7A-C shows expression of TMEM16E and TMEM16F in primary endothelial cells.
  • FIG. 8A-E shows validation of siRNA targeting TMEM16E and TMEM16F.
  • FIG. 8A HUVECs stably expressing TMEM16E containing a C-terminal V5 tag were transfected with indicated siRNA targeting TMEM16E or untargeted control siRNA for 72 h. TMEM16E protein was determined by SDS-PAGE and immunoblotting with antibodies against TMEM16E, V5, and actin (loading control). TMEM16E is detected as a band running just above 100 kd.
  • FIG. 8B Primary HUVECs were transfected with siRNA targeting TMEM16E for 72 h before determining TMEM16E mRNA level by quantitative PCR.
  • FIG. 8C C.
  • HUVECs were transfected with siRNA targeting TMEM16F for 72 h.
  • TMEM16F protein was determined by SDS- PAGE and immunoblotting with anti-TMEM16F antibody.
  • FIG. 8D Primary HUVECs or HUVECs stably expressing TMEM16E-V5 were transfected with indicated siRNA for 72 h prior to determination of TMEM16E and TMEM16F protein by SDS-PAGE and immunoblotting with anti-TMEM16F and anti-V5 antibody.
  • FIG. 8E HUVECs were transfected with indicated siRNA for 72 h and cell viability was determined by XTT assay. #1 and #2 denote independent siRNA sequences. TF denotes siRNA targeting tissue factor.
  • FIG. 9 shows that TMEM16E and TMEM16F regulate tissue factor procoagulant activity.
  • An Ea.hy926 cell line stably expressing tissue factor was transfected with siRNAs targeting TMEM16E, TMEM16F, or TF for 72 h. Cells were treated with Ca 2+ ionophore A23187 (6 pM) for 20 min and assayed for their ability to support factor Vila catalyzed conversion of factor X to factor Xa.
  • 16E, 16F and TF denote siRNA targeting TMEM16E, TMEM16F, and tissue factor, respectively.
  • #1 and #2 denote independent siRNA sequences. Error bars indicate mean ⁇ SD, ANOVA with Tukey’s posttest, ****p ⁇ 0.0001.
  • FIG. 10A-C shows that TMEM16E and TMEM16F are required for PS extemalization on ECs.
  • HUVECs were transfected with indicated siRNAs for 72 h, stimulated with TNF-a (10 ng/mL) for 18 h (FIG. 10A) or calcium ionophore A23187 (6 pM) for 20 min (FIG. 10B), and stained with annexin V to detect PS extemalization and Zombie Red to detect cell death. Each dot represents the total fluorescent area of Zombie Red per image normalized to the number of nuclei present. Note no increase in cell death (Zombie Red positivity) in cells treated with TNF-a or A23187 compared to control.
  • FIG. 10A-C shows that TMEM16E and TMEM16F are required for PS extemalization on ECs.
  • HUVECs were transfected with indicated siRNAs for 72 h, stimulated with TNF-a (10 ng/mL)
  • FIG. 11A-C shows phosphatidylserine externalization during thrombus formation following laser injury. Thrombus formation was monitored for 180 seconds in wild-type mice following laser injury of the cremasteric arteriole (FIG. 11A) and additionally in the presence of the platelet aggregation inhibitor eptifibatide (FIG. 11B, 10 pg/g of body weight). Representative images at indicated time points of the PS probe annexin V (red, Alexa Fluor 647), platelets (anti- CD42b antibody, blue, Dylight 405), and fibrin (anti-fibrin antibody, green, Dylight 488). Note annexin V positivity on the vessel wall in the absence of platelet aggregation.
  • FIG. 11A Asterisk (*) indicates the platelet aggregate.
  • FIG. 11C Annexin V staining following laser ablation. Annexin V is often observed wrapping around the vessel to the opposite wall. Dotted yellow lines indicate the vessel wall boundaries.
  • FIG. 11D 3-dimensional renderings of Z-stack images of annexin V binding following laser injury from the image in FIG. 11C. Scale bar is 25 pm unless otherwise indicated. [0022] FIG.
  • FIG. 12A-E shows that annexin V inhibits thrombosis. Thrombus formation was monitored for 180 seconds in wild-type mice following laser injury of the cremasteric arteriole in the presence or absence of annexin V (0.025 pg/g of body weight). Platelet and fibrin accumulation were monitored by anti-CD42b and anti-fibrin antibody conjugated to Dylight 647 and 488, respectively. Kinetics and magnitude of median integrated relative fluorescent units (RFU) for platelet (FIG. 12A) and fibrin (FIG. 12C) accumulation are shown following laser injury. The area under the curve (AUC) for fluorescent intensity was determined for platelets (FIG. 12B), fibrin (FIG. 12D) for each thrombus, analyzed by Mann-Whitney. FIG. 12E. Injury sizes associated with the thrombi analyzed above, analyzed by Student’s t test.
  • RNU median integrated relative fluorescent units
  • FIG. 13A-E shows blood and coagulation parameters in TMEM16E" ' mice.
  • Blood from TMEM16E’ ’ (AHO5' ⁇ ) and TMEM16 +/+ (Ano5 ) littermate controls was assessed for white blood cells (WBC, FIG. 13A), hemoglobin (Hg, FIG. 13B), and platelet counts (FIG. 13C).
  • FIG. 14A -C shows correlation of annexin V binding with vessel wall injury size.
  • Embodiments provided herein include a method of treating a thrombotic disorder, including administering a therapeutically effective amount of one or more TMEM16 inhibitors to a patient in need thereof.
  • the TMEM16 is TMEM16E, TMEM16F, or any combination thereof.
  • the one or more TMEM16 inhibitors is benzbromarone, isoquercetin, quercetin aglycone, quercetin derivatives, quercetin-3-O-glucoside (also known as isoquercetin), quercetin-5-O-glucoside, quercetin-7-O-glucoside, quercetin-9-O- glucoside quercetin-3’-O-glucoside, quercetin-4’-O-glucoside, quercetin-3-O-rutinoside (also known as rutin), quercetin-3-O-[a-rhamnosyl- (l->2)-a-rhamnosyl -(l->6)]-I3-glucoside, quercetin-3-O-galactoside, quercetin-7-O-galactoside, quercetin-3-O-rhamnoside, quercetin-7-O- galactoside, quercetin-glyco
  • the one or more TMEM16 inhibitors include benzbromarone and isoquercetin, and administering benzbromarone and isoquercetin results in a synergistic antithrombotic effect.
  • Additional embodiments of the method provided herein include also administering zafirlukast.
  • administering one or more TMEM16 inhibitors, and additionally administering zafirlukast results in a synergistic antithrombotic effect.
  • a therapeutically effective amount of benzbromarone is from about 50 mg to about 800 mg per day. In some embodiments of the method described herein, a therapeutically effective plasma concentration of benzbromarone is from about 1 to about 30 micromolar. In certain embodiments, a therapeutically effective amount of benzbromarone is about 5 pg/g. In some embodiments, the therapeutically effective amount of benzbromarone is administered between about every 12 to about every 24 hours. In some embodiments, the therapeutically effective amount of benzbromarone is administered about every 24 hours.
  • the thrombotic disorder is a thrombotic disease, hypercoagulation, blood coagulation, TMEM16-induced coagulation, coronary artery disease, cardiovascular disease, procoagulant endothelial cell dysfunction, procoagulant pulmonary epithelial cell dysfunction, aberrant procoagulant activity, pathological phosphoserine extemalization, dysregulated blood coagulation, antiphospholipid antibody syndrome, SARS-CoV-2 pneumonia, thrombosis during severe SARS-CoV-2 infection, post-acute sequelae of SARS CoV-2 infection, cancer-associated thrombosis, refractory thrombosis, myocardial infarction, stroke, venous thromboembolism, disseminated intravascular coagulation (DIC), sepsis, paroxysmal nocturnal hemoglobinuria, antiphospholipid antibody syndrome, myeloproliferative disorders, inherited red cell disorders associated with thrombosis, sickle cell disease,
  • DIC disseminated intravascular coagulation
  • the method relates to endothelial cells, epithelial cells, cancer cells, or other cells that can be involved in thrombotic diseases.
  • administering the therapeutically effective amount of one or more TMEM16 inhibitors does not increase bleeding complications, bleeding time, bleeding risk, or any combination thereof.
  • administering the therapeutically effective amount of one or more TMEM16 inhibitors results in a reduction in at least one indicator of thrombotic disease, results in at least one indicator of thrombotic disease remaining within an acceptable range, or a combination thereof.
  • the at least one indicator of thrombotic disease is D-dimer, thrombin antithrombin complex, prothrombin activation peptide Fl+2, procoagulant extracellular vesicle TF cofactor activity, factor Xa generation, thrombin generation, or plasma endogenous thrombin potential.
  • administering the therapeutically effective amount of one or more TMEM16 inhibitors results in concentrations of D-dimer in the patient remaining below about 500 ng/mL.
  • the one or more TMEM16 inhibitors is administered in combination with a blood-thinning medication, chemotherapy, or a drug indicated for the treatment of heart disease.
  • the one or more TMEM16 inhibitors is administered in combination with warfarin, heparin, low molecular weight heparins, ultra-low molecular weight heparins, direct oral anticoagulants targeting thrombin, factor Xa, factor Xia, parenteral direct thrombin inhibitors, argatroban, aspirin, P2Y12 inhibitors, clopidogrel, or a combination thereof.
  • the thrombotic disorder is associated with COVID- 19.
  • the patient has cancer.
  • the cancer is multiple myeloma, hematologic cancer, adenocarcinoma, cancer of the pancreas, stomach, ovaries, prostate, colon, lung, brain, breast, kidney, skin, cervix, or ear-nose-throat cancer.
  • FIG. 1 Further embodiments of the method provided herein are directed to a method for treating hypercoagulation related to COVID-19, including administering a therapeutically effective amount of one or more TMEM16 inhibitors to a patient in need thereof.
  • the COVID-19 is post-acute sequelae of SARS CoV-2 infection.
  • the TMEM16 is TMEM16E, TMEM16F, or any combination thereof.
  • the one or more TMEM16 inhibitors is benzbromarone, isoquercetin, ivermectin, crofelemer, nitazoxanide, hexachlorophene, dichlorophen, nitazoxanide, trifluoperazine, or a combination thereof.
  • the one or more TMEM16 inhibitors includes benzbromarone.
  • the one or more TMEM16 inhibitors further includes isoquercetin.
  • administering the one or more TMEM16 inhibitors including benzbromarone and isoquercetin results in a synergistic antihypercoagulation effect.
  • a therapeutically effective amount of benzbromarone is from about 50 mg to about 800 mg per day. In some embodiments, a therapeutically effective plasma concentration of benzbromarone is from about 1 to about 30 micromolar. In some embodiments, a therapeutically effective amount of benzbromarone is about 5 pg/g. In some embodiments, the therapeutically effective amount of benzbromarone is administered between about every 12 to about every 24 hours. In certain embodiments, the therapeutically effective amount of benzbromarone is administered about every 24 hours.
  • the hypercoagulation related to COVID-19 is microcoagulation, microvascular thrombosis, immunothrombosis, TMEM16- induced coagulation, coronary artery disease, cardiovascular disease, procoagulant endothelial cell dysfunction, procoagulant pulmonary epithelial cell dysfunction, aberrant procoagulant activity, pathological phosphoserine externalization, dysregulated blood coagulation, antiphospholipid antibody syndrome, SARS-CoV-2 pneumonia, thrombosis during severe SARS-CoV-2 infection, refractory thrombosis, myocardial infarction, stroke, venous thromboembolism, disseminated intravascular coagulation (DIC), sepsis, paroxysmal nocturnal hemoglobinuria, antiphospholipid antibody syndrome, myeloproliferative disorders, acute coronary syndrome, atrial fibrillation, or any combination thereof.
  • DIC disseminated intravascular coagulation
  • Additional embodiments provide that administering the therapeutically effective amount of one or more TMEM16 inhibitors does not increase bleeding complications, bleeding time, bleeding risk, or any combination thereof.
  • administering the therapeutically effective amount of one or more TMEM16 inhibitors results in a reduction in at least one indicator of hypercoagulation, results in at least one indicator of hypercoagulation remaining within an acceptable range, or a combination thereof.
  • the at least one indicator of hypercoagulation is D- dimer, thrombin antithrombin complex, prothrombin activation peptide Fl+2, procoagulant extracellular vesicle TF cofactor activity, factor Xa generation, thrombin generation, or plasma endogenous thrombin potential.
  • administering the therapeutically effective amount of one or more TMEM16 inhibitors results in concentrations of D-dimer in the patient remaining below about 500 ng/mL.
  • the one or more TMEM16 inhibitors is administered in combination with oral antiviral drugs, intravenous antiviral drugs, monoclonal antibodies, bloodthinning medications, or a drug indicated for the treatment of heart disease.
  • the one or more TMEM 16 inhibitors is administered in combination with direct oral anticoagulants, dabigatran, rivaroxaban, apixaban, edoxaban, betrixaban, Paxlovid, molnupiravir, remdesivir, Bebtelovimab, Evusheld, warfarin, heparin, low molecular weight heparins, ultra-low molecular weight heparins, direct oral anticoagulants targeting thrombin, factor Xa, factor Xia, parenteral direct thrombin inhibitors, argatroban, aspirin, P2Y12 inhibitors, clopidogrel, or a combination thereof.
  • the method described herein also includes administering zafirlukast.
  • administering the one or more TMEM16 inhibitors results in a synergistic anti-hypercoagulation effect.
  • Further embodiments of the method provided herein include a method for reducing cancer-associated thrombosis, including administering to a patient a therapeutically effective amount of one or more TMEM16 inhibitors.
  • the TMEM16 is TMEM16E, TMEM16F, or any combination thereof.
  • the one or more TMEM16 inhibitors is benzbromarone, isoquercetin, ivermectin, crofelemer, nitazoxanide, hexachlorophene, dichlorophen, nitazoxanide, trifluoperazine, or a combination thereof.
  • the one or more TMEM16 inhibitors includes benzbromarone.
  • the one or more TMEM16 inhibitors further includes isoquercetin.
  • administering the one or more TMEM16 inhibitors including benzbromarone and isoquercetin results in a synergistic antithrombotic effect.
  • the method described herein further includes administering zafirlukast.
  • administering the one or more TMEM16 inhibitors with zafirlukast results in a synergistic antithrombotic effect.
  • a therapeutically effective amount of benzbromarone is from about 50 mg to about 800 mg per day. In some embodiments, a therapeutically effective plasma concentration of benzbromarone is from about 1 to about 30 micromolar. In some embodiments, a therapeutically effective amount of benzbromarone is about 5 pg/g. In some embodiments, the therapeutically effective amount of benzbromarone is administered between about every 12 to about every 24 hours. In certain embodiments, the therapeutically effective amount of benzbromarone is administered about every 24 hours.
  • the patient has a thrombotic disease, cardiovascular disease, procoagulant endothelial cell dysfunction, procoagulant pulmonary epithelial cell dysfunction, aberrant procoagulant activity, pathological phosphoserine extemalization, dysregulated blood coagulation, antiphospholipid antibody syndrome, cancer-associated thrombosis, refractory thrombosis, myocardial infarction, stroke, venous thromboembolism, disseminated intravascular coagulation (DIC), sepsis, paroxysmal nocturnal hemoglobinuria, antiphospholipid antibody syndrome, myeloproliferative disorders, inherited red cell disorders associated with thrombosis, sickle cell disease, hereditary xerocytosis, acute coronary syndrome, atrial fibrillation, or any combination thereof.
  • DIC disseminated intravascular coagulation
  • administering the therapeutically effective amount of one or more TMEM16 inhibitors does not increase bleeding complications, bleeding time, bleeding risk, or any combination thereof.
  • administering the therapeutically effective amount of one or more TMEM16 inhibitors results in a reduction in at least one indicator of thrombotic disease, results in at least one indicator of thrombotic disease remaining within an acceptable range, or a combination thereof.
  • the at least one indicator of thrombotic disease is D- dimer, thrombin antithrombin complex, prothrombin activation peptide Fl+2, procoagulant extracellular vesicle TF cofactor activity, factor Xa generation, thrombin generation, or plasma endogenous thrombin potential.
  • administering the therapeutically effective amount of one or more TMEM16 inhibitors results in concentrations of D-dimer in the patient remaining below about 500 ng/mL.
  • the one or more TMEM16 inhibitors is administered in combination with a blood-thinning medication, chemotherapy, or a drug indicated for the treatment of heart disease.
  • the one or more TMEM16 inhibitors is administered in combination with warfarin, heparin, low molecular weight heparins, ultra-low molecular weight heparins, direct oral anticoagulants targeting thrombin, factor Xa, factor Xia, parenteral direct thrombin inhibitors, argatroban, aspirin, P2Y12 inhibitors, clopidogrel, or a combination thereof.
  • the cancer is multiple myeloma, hematologic cancer, adenocarcinoma, cancer of the pancreas, stomach, ovaries, prostate, colon, lung, brain, breast, kidney, skin, cervix, or ear-nose-throat cancer.
  • compositions suitable to treat a thrombotic disorder including a therapeutically effective amount of one or more TMEM16 inhibitors, and pharmaceutically acceptable excipients.
  • the TMEM16 is TMEM16E, TMEM16F, or any combination thereof.
  • the one or more TMEM16 inhibitors is benzbromarone, isoquercetin, ivermectin, crofelemer, nitazoxanide, hexachlorophene, dichlorophen, nitazoxanide, trifluoperazine, or a combination thereof.
  • the one or more TMEM16 inhibitors includes benzbromarone.
  • the one or more TMEM16 inhibitors further includes isoquercetin.
  • administering the one or more TMEM16 inhibitors including benzbromarone and isoquercetin results in a synergistic antithrombotic effect.
  • the therapeutic composition described herein further includes administering zafirlukast.
  • administering the one or more TMEM16 inhibitors, and further including administering zafirlukast results in a synergistic antithrombotic effect.
  • a therapeutically effective amount of benzbromarone is from about 50 mg to about 800 mg per day. In some embodiments of the method described herein, a therapeutically effective plasma concentration of benzbromarone is from about 1 to about 30 micromolar. In certain embodiments, a therapeutically effective amount of benzbromarone is about 5 pg/g. In some embodiments, the therapeutically effective amount of benzbromarone is administered between about every 12 to about every 24 hours. In some embodiments, the therapeutically effective amount of benzbromarone is administered about every 24 hours.
  • the thrombotic disorder is a thrombotic disease, cardiovascular disease, procoagulant endothelial cell dysfunction, procoagulant pulmonary epithelial cell dysfunction, aberrant procoagulant activity, pathological phosphoserine extemalization, dysregulated blood coagulation, antiphospholipid antibody syndrome, SARS- CoV-2 pneumonia, thrombosis during severe SARS-CoV-2 infection, post-acute sequelae of SARS CoV-2 infection, cancer-associated thrombosis, refractory thrombosis, myocardial infarction, stroke, venous thromboembolism, disseminated intravascular coagulation (DIC), sepsis, paroxysmal nocturnal hemoglobinuria, antiphospholipid antibody syndrome, myeloproliferative disorders, inherited red cell disorders associated with thrombosis, sickle cell disease, hereditary xerocytosis, acute coronary syndrome, atrial fibrillation, or any combination thereof.
  • DIC disseminated intravascular
  • the therapeutically effective amount of one or more TMEM16 inhibitors does not increase bleeding complications, bleeding time, bleeding risk, or any combination thereof. [0061] In certain embodiments of the therapeutic composition provided herein, the therapeutically effective amount of one or more TMEM16 inhibitors results in a reduction in at least one indicator of thrombotic disease, results in at least one indicator of thrombotic disease remaining within an acceptable range, or a combination thereof.
  • the at least one indicator of thrombotic disease is D-dimer, thrombin antithrombin complex, prothrombin activation peptide Fl+2, procoagulant extracellular vesicle TF cofactor activity, factor Xa generation, thrombin generation, or plasma endogenous thrombin potential.
  • the therapeutically effective amount of one or more TMEM16 inhibitors results in concentrations of D-dimer in a patient remaining below about 500 ng/mL.
  • the term “about” means plus or minus 10% of the numerical value of the number with which it is being used. Therefore, about 50% means in the range of 45%-55%.
  • the terms “treat,” “treated,” or “treating” as used herein refers to both therapeutic treatment and prophylactic or preventative measures, wherein the object is to inhibit, prevent or slow down or reduce the full effect or likelihood of (lessen) any undesired physiological condition, disorder or disease, or to improve, inhibit, or otherwise obtain beneficial or desired clinical results.
  • beneficial or desired clinical results include, but are not limited to, improvement or alleviation of symptoms; diminishment of the extent of the condition, disorder or disease; stabilization (i.e., not worsening) of the state of the condition, disorder or disease; delay in onset or slowing of the progression of the condition, disorder or disease; amelioration of the condition, disorder or disease state; and remission (whether partial or total), whether detectable or undetectable, or enhancement or improvement of the condition, disorder or disease.
  • Treatment includes eliciting a clinically significant response without excessive levels of side effects. Treatment also includes prolonging survival as compared to expected survival if not receiving treatment.
  • the term “effective amount” refers to an amount that results in measurable inhibition of at least one symptom or parameter of a specific disorder or pathological process.
  • the term “therapeutically effective amount” of compositions of the application is an amount which confers a therapeutic effect on the treated subject at a reasonable benefit/risk ratio applicable to any medical treatment.
  • the therapeutic effect may be objective (that is, measurable by some test or marker) or subjective (that is, subject gives an indication of or feels an effect, or physician observes a change).
  • terapéuticaally effective amount of an active agent means an amount effective, when administered to a patient, to provide a therapeutic benefit such as a prevention, inhibition, or an amelioration of symptoms, e.g., to prevent the activation of mast cells and prevent the formation of mast cell activated cytokines in a patient suffering from amyotrophic lateral sclerosis (ALS).
  • a therapeutically effective amount may vary according to factors such as the health, age, and weight of the patient, and the ability of the compound to elicit a desired response in the patient. Dosage regimens may be adjusted to provide the optimum therapeutic response.
  • a therapeutically effective amount is also one in which any toxic or detrimental effects (e.g., side effects) of the active agent are outweighed by the therapeutically beneficial effects.
  • the term “patient” as used herein includes, but is not limited to, humans and nonhuman vertebrates such as wild, domestic, and farm animals.
  • the subject described herein is an animal. In certain embodiments, the subject is a mammal. In certain embodiments, the subject is a human. In certain embodiments, the subject is a non-human animal.
  • the subject is a non-human mammal.
  • the subject is a domesticated animal, such as a dog, cat, cow, pig, horse, sheep, or goat.
  • the subject is a companion animal such as a dog or cat.
  • the subject is a livestock animal such as a cow, pig, horse, sheep, or goat.
  • the subject is a zoo animal.
  • the subject is a research animal such as a rodent, dog, or non-human primate.
  • the subject is a non-human transgenic animal such as a transgenic mouse or transgenic pig.
  • pharmaceutically acceptable excipients it is meant the carrier, diluent or excipient must be compatible with the other ingredients of the topical formulation and not deleterious to the recipient thereof.
  • thrombotic disorder refers to many distinct conditions that cause or increase the risk of a venous or arterial thrombotic event, including but not limited to, atrial fibrillation, thrombosis due to a mechanical heart valve, myocardial infarction, unstable angina, deep vein thrombosis, acute ischemic stroke, pulmonary embolism, atherosclerosis, factor V Leiden, antithrombin III deficiency, protein C deficiency, protein S deficiency, prothrombin gene mutation (G20210A), hyperhomocysteinemia, antiphospholipid antibody syndrome, anticardiolipin antibody, thrombosis syndrome, lupus anticoagulant syndrome, malignancy, major surgery, immobilization, oral contraceptive use, thalidomide use, especially in combination with dexamethasone, heparin-induced thrombocytopenia, pregnancy, myeloproliferative disorders, inflammatory bowel disease, nephrotic syndrome, paroxysmal
  • thrombotic disorder also refers to thrombosis induced by cancer, e.g., multiple myeloma and other hematologic cancers, adenocarcinoma, cancer of the pancreas, stomach, ovaries, prostate, colon, lung, brain, breast, kidney, skin, cervix, and ear- nose- throat cancer.
  • cancer e.g., multiple myeloma and other hematologic cancers, adenocarcinoma, cancer of the pancreas, stomach, ovaries, prostate, colon, lung, brain, breast, kidney, skin, cervix, and ear- nose- throat cancer.
  • blood thinning medication refers to an antiplatelet drug, e.g., clopidogrel bisulfate, heparin, warfarin, enoxaparin, abciximab, eptifibatide, tirofiban, prasugrel, ticlopidine, beraprost, prostacyclin, iloprost, treprostinil, aspirin, aloxiprin, carbasalate calcium, indobufen, triflusal, dipyridamole, picotamide, terutroban, cilostazol, cloricromen, ditazole; or an anticoagulant, e.g., acenocoumarol, coumatetralyl, dicoumarol, ethyl biscoumacetate, phenprocoumon, clorindione, diphenadione, phenindione, tioclomarol, bemiparin
  • an antiplatelet drug
  • isoquercetin refers to certain active compounds for administration as described herein.
  • Isoquercetin (2-(3,4-Dihydroxyphenyl)-5,7-dihydroxy-3-[(2S,3R,4S,5S,6R)-3,4,5- trihydroxy-6-(hydroxymethyl)oxan-2-yl]oxychromen-4-one) is a 3-O-glucoside of quercetin having the following structure:
  • Quercetin is characterized by the following structure:
  • the active compound may encompass quercetin or quercetin derivatives such as: quercetin-5-O-glucoside, quercetin-7-O-glucoside, quercetin-9- O-glucoside, quercetin-3-O-[.alpha.-rhamnosyl-(l ,fwdarw.2)-.alpha.-rhamnosyl-(l .fwdarw- .6)]- . beta.
  • quercetin or quercetin derivatives such as: quercetin-5-O-glucoside, quercetin-7-O-glucoside, quercetin-9- O-glucoside, quercetin-3-O-[.alpha.-rhamnosyl-(l ,fwdarw.2)-.alpha.-rhamnosyl-(l .fwdarw- .6)]- . beta.
  • quercetin derivatives are converted in the body to quercetin aglycon and/or other active derivatives, including methylated, sulphated and gluconorated forms which are absorbed in the body.
  • the quercetin or quercetin derivative can be added to the composition either in a pure form or as an ingredient in a mixture (e.g., a plant extract).
  • examples of commercially available quercetin include QU995 (containing 99.5% quercetin) and QU985 (containing 98.5% quercetin) from Quercegen Pharmaceuticals LLC (Boston, Mass.).
  • one of the TMEM16 inhibitors for use in the present methods is isoquercetin.
  • Isoquercetin can also be quercetin or derivatives of isoquercetin.
  • the isoquercetin derivative can be added to the composition either in a pure form or as an ingredient in a mixture (e.g., a plant extract).
  • examples of commercially available isoquercetin compounds include those available from Quercegen Pharmaceuticals LLC: ISQ 995 AN (99.5% pure all-natural isoquercetin) and ISQ 995 CIT (99.5% pure isoquercitrin). Additional methods and isoquercetin compositions can be found in U.S. Pat. Nos. 7,745,486 and 7,745,487, incorporated herein by reference.
  • the therapeutic composition of at least two TMEM16 inhibitors may include benzbromarone, isoquercetin, zafirlukast, or any combination thereof.
  • the therapeutic composition may be administered by oral or parenteral (e.g., intramuscular, intraperitoneal, intravenous, ICV, intracistemal injection or infusion, subcutaneous injection, or implant) dosage form and may be formulated, alone or together, in suitable dosage unit formulations containing conventional non-toxic pharmaceutically acceptable carriers, adjuvants and vehicles appropriate for each route of administration.
  • the compounds and compositions described herein may also be formulated as a controlled-release formulation.
  • the therapeutic composition can be administered in a wide range of dosage-forms including, for example, solid dosage forms and liquid dosage forms.
  • Solid dosage forms may include powders, tablets, pills, capsules, suppositories, or dispersible granules.
  • a solid carrier can be one or more substances that function as a diluting agent, flavor additive, solvent, lubricant, suspension agent, binder, preservative, tablet-disintegrating substance or encapsulating material.
  • the carrier may be a finely pulverized solid including lactose, hydroxypropyl methylcellulose and PVP, mixed with an appropriate amount of the active ingredient.
  • Appropriate carriers for powder and tablet forms include for example magnesium carbonate, magnesium stearate, talc, sugar, lactose, pectin, dextrin, stiffeners, gelatins, tragacanth, methylcellulose, and sodium carboxymethylcellulose.
  • Liquid dosage forms include for example solutions, suspensions, and emulsions. Also included are compositions in solid form that are meant to be converted to liquid form shortly prior to consumption. These forms may include, in addition to the active ingredients, artificial colors, flavors, stabilizers, buffers, natural or artificial sweeteners, dispersing agents, thickeners, dissolving agents and the like.
  • Solutions or mixtures may be administered directly to the nasal cavity using conventional means, such as drops or sprays.
  • the composition may be produced in individual or multi-dose forms. Multi-dose forms would include a dropper, pipette or atomizer that delivers a predetermined volume of the composition.
  • the therapeutic composition may be provided in individual dosage units that contain a suitable amount of the active ingredient.
  • the individual doses may be provided in a package, or as a kit that includes a measuring device, e.g., a device for measuring oral or injectable dosages (i.e., a measuring cup, needle, or syringe).
  • a measuring device e.g., a device for measuring oral or injectable dosages (i.e., a measuring cup, needle, or syringe).
  • the kit can also include, other materials such buffers, diluents, filters, and package inserts with instructions for use.
  • a label may be present on the on the kit to indicate that the composition is used for a specific therapy, and may also indicate directions for use.
  • the therapeutic compositions of the present invention may further comprise one or more additional active agents.
  • any of the active agents may be administered in the form of the compound per se, and/or in the form of a salt, polymorph, ester, amide, prodrug, derivative, or the like, provided the salt, polymorph, ester, amide, prodrug or derivative is suitable pharmacologically.
  • salts, esters, amides, prodrugs and other derivatives of the active agents may be prepared using standard procedures known to those skilled in the art of synthetic organic chemistry and described, for example, by J. March, Advanced Organic Chemistry: Reactions, Mechanisms and Structure, 4th Ed. (New York: Wiley- Interscience, 1992).
  • the active agent may be incorporated into the present compositions either as the racemate or in enantiomerically enriched form.
  • the dosage of the therapeutic composition, according to any embodiment described being administered will depend on the condition being treated, the particular compound, and other clinical factors such as age, sex, weight, and health of the subject being treated, the route of administration of the compound(s), and the type of composition being administered (tablet, gel cap, capsule, solution, suspension, inhaler, aerosol, elixir, lozenge, injection, patch, ointment, cream, etc.). It is to be understood that the present disclosure has application for both human and animal use. The amount of the therapeutic composition according to any embodiment described, required for use in treatment will be ultimately at the discretion of the attendant physician or clinician.
  • the therapeutic composition administered in the methods of this invention can be a sterile injectable or infusible solution that contains the therapeutic composition together with pharmaceutically acceptable excipients.
  • Pharmaceutically acceptable excipients may include, but are not limited to, buffering agents, coloring agents, diluents, di si nt egrants, emulsifiers, flavorants, glidants, lubricants, preservatives, stabilizers, surfactants, tableting agents, and wetting agents.
  • Some excipients may be listed in more than one class, for example vegetable oil may be used as a lubricant in some formulations and a diluent in others.
  • Exemplary pharmaceutically acceptable excipients include sugars, starches, celluloses, powdered tragacanth, malt, gelatin, talc, and vegetable oils.
  • Optional additional active agents may be included in a pharmaceutical composition, which do not substantially interfere with the activity of the active agent.
  • the amount of excipient employed in conjunction with the compound is sufficient to provide a practical quantity of material for administration per unit dose of the active agent.
  • the dosage of the active ingredients in the therapeutic compositions of this invention may be varied so that a suitable dosage form is obtained.
  • the active ingredients may be administered to patients (animals and human) in need of such treatment in dosages that will provide optimal pharmaceutical efficacy.
  • the selected dosage depends upon the desired therapeutic effect, on the route of administration, and on the duration of the treatment.
  • the dose will vary from patient to patient depending upon the nature and severity of disease, the patient's weight, special diets then being followed by a patient, concurrent medication, and other factors which those skilled in the art will recognize.
  • the therapeutically effective amount of benzbromarone will be about 50 mg to about 800 mg daily. In certain additional embodiments, the therapeutically effective amount will be about 50 mg, 100 mg, or 200 mg daily. In certain embodiments, the therapeutically effective amount will be about 50 mg to about 500 mg daily.
  • the therapeutically effective amount of isoquercetin is between a lower limit of about 50 mg/day, about 52 mg/day, about 55 mg/day, about 57 mg/day, about 60 mg/day, about 62 mg/day, about 65 mg/day, about 67 mg/day, about 70 mg/day, about 72 mg/day, about 75 mg/day, about 77 mg/day, about 80 mg/day, about 82 mg/day, about 85 mg/day, about 87 mg/day, about 90 mg/day, about 92 mg/day, about 95 mg/day, about 97 mg/day, about 100 mg/day, about 102 mg/day, about 105 mg/day, about 107 mg/day, about 110 mg/day, 112 mg/day, about 115 mg/day, about 117 mg/day, about 120 mg/day, 122 mg/day, about 125 mg/day, about 127 mg/day, about 130 mg/day, 132 mg/day, about 135 mg/day, about 50 mg/day, about 52 mg
  • the therapeutically effective amount of isoquercetin will be about 500 mg to up to 5 grams daily. In certain additional embodiments, the therapeutically effective amount will be about 4 grams, or 3 grams, or even 2 grams. In certain embodiments, the therapeutically effective amount will be about 500 mg to about 2000 mg daily.
  • the therapeutically effective amount of isoquercetin is between a lower limit of about 500 mg/day, about 525 mg/day, about 550 mg/day, about 575 mg/day, about 600 mg/day, about 625 mg/day, about 650 mg/day, about 675 mg/day, about 700 mg/day, about 725 mg/day, about 750 mg/day, about 775 mg/day, about 800 mg/day, about 825 mg/day, about 850 mg/day, about 875 mg/day, about 900 mg/day, about 925 mg/day, about 950 mg/day, about 975 mg/day, about 1000 mg/day, about 1025 mg/day, about 1050 mg/day, about
  • the compounds may be administered on a regimen of 1 to 4 times per day, such as once, twice, three times or four times per day.
  • the coronavirus that causes COVID-19 can lead to hypercoagulation in a patient due to inflammation in endothelial and epithelial tissue.
  • the tissue affected can be in the lung, heart, veins, arteries, or other tissues.
  • Thrombosis related to COVID-19 can be detected as pulmonary embolism, large- vessel thrombosis, stroke, thromboses in blood vessels that supply the small intestine, obstructions in peripheral and large vessel arteries, and cerebral venous sinus thrombosis.
  • Microvascular inflammation and subsequent microthrombosis can lead to tissue damage, with long-term negative effects.
  • COVID-19 related hypercoagulation may be related to many physiological responses, including complement activation, the renin-angiotensin system, platelet activation, or a cytokine storm.
  • Treatment of hypercoagulation in COVID-19 commonly uses doses of heparin; the present disclosure provides a method of treating COVID- 19 related thrombosis or hypercoagulation with TMEM16 inhibitors, such as benzbromarone.
  • TMEM16 inhibitors such as benzbromarone.
  • when benzbromarone is administered in combination with isoquercetin it can produce a synergistic effect, including but not limited to, increased protection against micro and macro thrombosis, decreased platelet aggregation, decreased fibrin formation, and lowered levels of P-dimer and other indicators of thrombosis.
  • Post-acute sequelae of SARS CoV-2 infection, or long COVID occurs when a patient does not recover within the usual one or two weeks after contracting COVID-19.
  • Long COVID leads to a wide variety of symptoms, with multiorgan effects, and is difficult to diagnose and treat. There is an increased propensity for hypercoagulation in long COVID, and more treatments are needed to prevent the increased risks of damage to lungs, kidneys, and blood vessels.
  • Treatment with TMEM16 inhibitors is useful in both preventing and lowering the risk of thrombosis in patients with long COVID, and can be accomplished through administering benzbromarone or through administering a combination of benzbromarone with one or more of isoquercetin and zafirlukast.
  • VTE Venous thromboembolism
  • the types of cancer can be selected from the group consisting of estrogen receptor-dependent breast cancer, estrogen receptor-independent breast cancer, hormone receptor-dependent prostate cancer, hormone receptor-independent prostate cancer, brain cancer, renal cancer, glioblastoma, colon cancer, familial adenomatous polyposis (FAP), colorectal cancer, pancreatic cancer, bladder cancer, esophageal cancer, stomach cancer, genitourinary cancer, gastrointestinal cancer, uterine cancer, ovarian cancer, astrocytomas, gliomas, skin cancer, squamous cell carcinoma, Keratoakantoma, Bowen disease, cutaneous T-Cell Lymphoma, melanoma, basal cell carcinoma, actinic keratosis; ichtiosis; acne, acne vulgaris, sarcomas, Kaposi's sarcoma, osteosarcoma, head and neck cancer, small cell lung carcinoma, non-small cell lung carcinoma, leukemia,
  • Additional cancers that will benefit from the methods described herein include cancers associated with certain viruses (and include improving a pre-cancerous condition during viral infection).
  • Such conditions include those associated with Human T-cell leukemia virus type, also called human T-lymphotrophic virus (HTLV-1) which is linked to adult T-cell leukemia/lymphoma.
  • Another such cancer include those associated with human papillomavirus (HPV), which has at least 12 strains that can cause cancer in men and women, including anal, cervical, penile, throat, vaginal and vulvar cancer.
  • Additional condition includes those associated with human herpes virus 8 (HHV-8), which is associated with Kaposi sarcoma in people who have a weakened immune system (e.g. patients with HIV).
  • HIV-associated cancers include Kaposi sarcoma, non-Hodgkin’s and Hodgkin’s lymphoma, cervical cancer, and cancers of the anus, liver, mouth and throat and lung.
  • hepatitis C is a leading cause of liver cancer, and can cause non-Hodgkin’s lymphoma, and as such can benefit from the methods described herein.
  • hepatitis B is a leading cause of liver cancer, and these conditions can benefit from the methods described herein.
  • the cancer is a metastasizing cancer.
  • a metastasizing cancer is a cancer which may form or often forms metastases.
  • a metastasizing cancer which has already spread from the part of the body where it started, i.e. the primary site, to other parts of the body, is also denoted metastatic cancer.
  • metastatic cancer When cancer cells break away from a tumor, they can travel to other areas of the body through the bloodstream or the lymph system. Such cancer cells may then form new tumors in other areas of the body.
  • the cancer is a metastasizing cancer selected from the group consisting of metastasizing forms of Hodgkin lymphoma, colorectal cancer, cervical cancer, lung cancer, skin cancer such as squamous cell cancer or basal cell carcinoma, head and neck cancer, gastric cancer, pancreatic cancer, head and neck squamous cell cancer, and breast cancer.
  • the metastatic cancer is colorectal cancer, pancreatic cancer, or non-small cell lung cancer.
  • the cancer is classifiable as Stage III or Stage IV according to the TNM anatomic/prognostic group system of the cancer staging system of the American Joint Committee on Cancer. In additional embodiments, the cancer is classifiable as Stage IV according to the TNM anatomic/prognostic group system of the cancer staging system of the American Joint Committee on Cancer.
  • the cancer is a metastasizing cancer selected from the group consisting of metastasizing forms of Hodgkin lymphoma, colorectal cancer, cervical cancer, lung cancer, skin cancer such as squamous cell cancer or basal cell carcinoma, head and neck cancer, gastric cancer, pancreatic cancer, and breast cancer, wherein said metastasizing cancer is classifiable as Stage IV according to the TNM anatomic/prognostic group system of the cancer staging system of the American Joint Committee on Cancer (7.sup.th edition, 2010, Springer).
  • EVA.hy296 cells stably expressing tissue factor (EA.hy.296-TF high cells) (28) were cultured in DMEM containing 10% FBS.
  • siRNA Cells were reverse transfected with siRNA at a final concentration of 20 pM (96-well plate) or 40 pM (384-well plate) using Lipofectamine RNAiMax (ThermoFisher Scientific) according to manufacturer protocol. The following siRNA sequences were used (Dharmacon siGENOME or ON-TARGET) TMEM16E (ANO5y.
  • TMEM16F ANO6 GAUCAUCGCUUCAGUUAUU, CAACUCAGCUGACAAUAAU, TF (F3) CAUUGGAGCUGUGGUAUUU; Stiml (STIM1) siGENOME SMARTpool siRNA: CAUCAGAAGUAUACAAUUG, AGAAGGAGCUAGAAUCUCA,
  • tumor necrosis factor-a (TNF-a, 10 ng/mL, R&D Systems) for 3.5 hours and/or Ca 2+ ionophore A23187 (Sigma) for 20 min at 37°C, washed twice with HBS- BSA (20 mM HEPES, pH 7.4, 150 mM NaCl, 5 mM KC1, 5 mM CaCh, 1 mg/mL fatty acid-free bovine serum albumin, Sigma) and equilibrated to room temperature.
  • HBS- BSA 20 mM HEPES, pH 7.4, 150 mM NaCl, 5 mM KC1, 5 mM CaCh, 1 mg/mL fatty acid-free bovine serum albumin, Sigma
  • Factor Xa generation experiments cells were incubated with HBS-BSA containing factor X (125 nM, Haematologic Technologies, factor Vila (0.6 nM, Haematologic Technologies), and factor Xa chromogenic substrate Biophen-CSl 1(22) (150 pM, AniaraDiagnostica). Absorbance at 405 nm was measured every minute for 3 hours on an xMark Spectrophotometer (Bio-Rad). Maximal reaction velocity was converted to factor Xa nM/min based on standard curve analysis of isolated factor Xa (Haematologic Technologies) serial dilutions with the same reaction conditions. In the focused siRNA screen of PLSs (FIG.
  • cells were transfected with the corresponding Dharmacon siGENOME gene-specific siRNA pool in 384-well format and assayed as in the above kinetic experiments, except that factor Xa generated was determined at 45 minutes on an EnVision plate reader (Perkin Elmer) and presented as a percentage of untargeted siRNA control.
  • drug 0.03-30 pM
  • DMSO vehicle control was added at the time of TNF-a stimulation.
  • TF inhibitory antibody (10 pg/mL, 4509, BioMedica Diagnostics) or bovine lactadherin (100 nM, Haematologic Technologies) were evaluated, cells were washed in HBS-BSA and incubated in the presence of indicated protein in HBS-BSA for 10 min. The reaction was then triggered by addition of factors X and Vila and factor Xa subtrate at concentrations indicated above.
  • thrombin generation experiments cells were washed twice with HBS-BSA and incubated in 80 pL HBS plus 20 pL pooled human plasma (George King Bio-Medical) to supply coagulation factors and 5 mM H-Gly-Pro-Arg-Pro-OH fibrin polymerization inhibitor (GPRP, 5 mM, Cayman Chemical) and fluorogenic thrombin substrate B0C-L-FPR-ANSNH-C2H5 (50 pM, SN-20, Haematologic Technologies).
  • bovine lactadherin 100 pM, Haematologic Technologies was added to the reaction mixutre.
  • the reaction was initiated by addition of 0.8-1.0 mM CaCh and read immediately using a Synergy HTX plate reader (BioTek). Fluorescence (excitation 352 nm / emission 470 nm) was measured every minute for 1 hour. First derivative of thrombin generation curves were compared with a standard curve of thrombin to determine thrombin generated in U/mL. All individual experiments were performed in technical triplicate and the mean of these technical replicates was used as the value for each independent experiment.
  • HUVECs were plated directly in gelatin-coated glass chamber slides for 72 hours.
  • Cells were washed with 10 mM HEPES buffer (pH 7.4) containing 140 mM NaCl, 2.5 mM CaCh, (annexin V binding buffer) and stained with annexin V Alexa Fluor 488 (Thermo Fischer Scientific) at a 1:50 dilution and Zombie Red viability dye (BioLegend) at a 1 : 1000 dilution in the dark for 15 minutes at room temperature in annexin V binding buffer. Cells were washed and fixed in annexin V binding buffer containing 4% paraformaldehyde for 7 minutes.
  • telomere length was measured by telomere ⁇ telomere ⁇ telomere ⁇ telomere ⁇ telomere ⁇ telomere ⁇ telomere ⁇ telomere ⁇ telomere ⁇ telomere ⁇ telomere ⁇ telomere ⁇ telomere ⁇
  • HUVECs were stimulated with indicated concentration of Ca 2+ ionophore A23187 (Sigma) for 15 minutes at 37°C and washed with PBS.
  • Ca 2+ ionophore A23187 Sigma
  • drug 10 pM
  • DMSO vehicle control was added 90 minutes prior to A23187 stimulation.
  • Cells were dissociated using Accutase (StemCell Technologies), and washed by re-pelleting twice in cold PBS.
  • Confluent HUVECs were washed with HBSS (140 mM NaCl, 5 mM KC1, 1 mM CaCL, 4 mM MgSO4, 5 mM MgCE, 3 mM Na2HPO4, 4 mM KH2PO4, 6 mM D-glucose, 4 mM NaHCCf) and loaded with 100 pL of 4 pM Calbryte 520 AM (AAT Bioquest) in HBSS for 30 minutes. Calbryte 520 does not bind Ca 2+ until it is esterified intracellularly and therefore can be loaded in buffers containing divalent cations.
  • HBSS 140 mM NaCl, 5 mM KC1, 1 mM CaCL, 4 mM MgSO4, 5 mM MgCE, 3 mM Na2HPO4, 4 mM KH2PO4, 6 mM D-glucose, 4 mM NaHCCf
  • Hematologic analysis of whole blood counts was performed using a Hemavet 850FS (Drew Scientific) for white blood cell counts, hemoglobin measurement, and platelet counts.
  • the prothrombin time was measured using the Neoplastine Cl Plus (Diagnostica Stago, Inc.) and the activated partial thromboplastin time was measured using STA-PTT (Diagnostica Stago, Inc.).
  • Coagulation assays were read on a Start benchtop analyzer (Diagnostica Stago, Inc.) according to manufacturer’s instructions.
  • Gene expression was determined using a 2-step Cell-to-CT kit (Thermo Fisher Scientific) with the Hsl381106_ml (AN05), Hs03805835_ml (AN06), Hs00372436_sl and Hs00194899_ml (ACTB) gene expression probes (Taqman, Thermo Fisher Scientific). Quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) was performed in technical duplicate for each biologic sample using a QuantStudio 6 Flex real-time PCR system. Gene expression was compared to ACTB expression using the DDCt method.
  • TMEM16E (AN05) coding sequence was subcloned into the lentiviral vector pLX304 (Addgene plasmid #25890, gift from David Root).
  • Lenti-X 293T cells (Takara) were cotransfected with ANO5-pLX304 lentiviral transfer vector and packaging vectors (pMD2.G and psPAX2, Addgene plasmids #12259 and #12260, gift from Didier Trono) using Lipofectamine 3000 (ThermoFisher Scientific).
  • the viral supernatant was collected twice every 24 hours and replaced with fresh DMEM supplemented with 10% fetal bovine serum.
  • Primary HUVECs (passage 1-2) were cultured in the presence of viral supernatant for 6 hours and then replaced with complete endothelial cell growth media (see Endothelial cell culture and siRNA transfection) for 48 hours before selecting for transduced cells with addition of blasticidin (10 pg/mL) to the cell culture media.
  • HUVEC cultures were lysed using RIPA buffer (Boston Bioproducts) supplemented with cOmplete Protease Inhibitor Cocktail (Roche) and PhosStop phosphatase inhibitor cocktail (Roche), 1 mM Na3VO4 (New England Biolabs), and 1 mM NaF. Proteins were resolved via SDS-PAGE 4-12% gradient gels (Thermofisher Scientific) under reducing conditions using NuPAGE SDS sample buffer and sample reducing agent (ThermoFisher Scientific), transferred to a nitrocellulose membrane, and blocked with SuperBlock buffer (ThermoFisher Scientific).
  • Protein detection was performed with the following primary antibodies: TMEM16E/Ano5 (clone N421A/85, UC Davis/NIHNeuroMab), V5-Tag (#80076, Cell Signaling Technology), TMEM16F (Sigma), b-actin (#12620, Cell Signaling Technologies) and appropriate species-specific HRP-conjugated secondary antibodies (Cell Signaling Technologies). Immunoblots were developed with Supersignal West Dura or Femto Chemiluminescent Substrate (ThermoFisher Scientific) and visualized with Syngene BioImage and GeneSnap image acquisition software.
  • Wild-type C57BL/6J male mice were anesthetized and prepped as described in “Intravital microscopy and laser-induced vessel wall injury model” and the cremaster arteriole was injured with a laser ablation with (Ablate!, 3i).
  • Z-stack images were obtained on an CSU-W1 spinning disk confocal microscope with SoRa super resolution optical unit (Yokagawa) using a 6-line laser illumination system (3i), 63X high numerical aperture water corrected lens (Zeiss) and Ocra Fusion BT sCMOS digital video camera (Hamamatsu).
  • Z-stack images were rendered into 3-dimensional images using using Slidebook version 6.0 (Intelligent Imaging Innovations).
  • Intravital microscopy and laser-induced vessel wall injury model Thrombus formation was visualized via intravital microscopy following laser- induced injury to the cremasteric arteriole in male mice as previously described. Platelets were detected using anti-CD42b antibody (0.1 mg/g body weight; Emfret Analytics) conjugated to Dylight 405 (Thermo Fisher Scientific), fibrin was detected with anti-fibrin antibody (0.5 mg/g body weight; clone 59D8) conjugated to Dylight 488 (Thermo Fisher Scientific), and PS extemalization was detected using annexin V (0.025 pg/g body weight) conjugated to Alexa Fluor 647 (Thermo Fisher Scientific) infused via internal jugular vein catheter.
  • Annexin V was re-dosed every 10-15 minutes due to its rapid metabolism.
  • lactadherin conjugated to FITC Haematologic Technologies
  • pSIVA Novus Biologicals
  • the cremasteric microcirculation was surgically exposed and injury to the cremasteric arteriole was stimulated with a MicroPoint Laser System (Photonics Instruments). Imaging was performed on a M205 FCA microscope (Leica) with LEDbased fluorescence light engine (SpectraX, Lumencor).
  • Integrated fluorescence intensity Sum Intensity of signal - (mean of the maximal background intensity x area of the signal). This calculation was performed for all frames in each thrombus and plotted versus time to provide the kinetics of thrombus formation. Area under the curve was calculated individual thrombi to evaluate for statistical significance (Higgins et al., J Clin Invest 128:1471-1484 (2016), Schulman et al., J Clin Invest 130:5302-5312 (2020). For experiments with eptifibatide, mice were injected intravenously with eptifibatide (10 pg/g body weight, Cayman Chemical) every 10-15 minutes.
  • the tail was immersed in saline prewarmed to 37°C for 2 minutes. The tail was then amputated 5 mm from the tip and immediately immersed back into 15 mL of warmed saline. The total bleeding time (including re-bleeding time) was recorded for 10 minutes. The amount of blood loss was quantitated by measuring hemoglobin content of the blood collected in the 15 mL test tube. The red blood cells were pelleted by centrifugation (300 g for 6 minutes). The pellet was lysed in 6 mL red blood cell lysis buffer and the amount of hemoglobin was spectrophotometrically determined by measuring light absorbance at 575 nm.
  • TMEM16E ⁇ nodj-null mice on a C57BL/6J background were described previously (33). Wild-type C57BL/6J mice were obtained from Jackson Laboratory (Bar Harbor, ME). Experiments were performed on mice of 8-12 weeks of age.
  • benzbromarone (Cayman Chemical) the compound was dissolved to a concentration of 40 pg/mL in DMSO and dissolved to a working solution in com oil to a final concentration of 1 pg/mL.
  • Benzbromarone or DSMO vehicle control in corn oil was injected intraperitoneally at a concentration of 5 ug/g body weight 1 hour prior to experiments.
  • mice were anesthetized with intraperitoneal injection of ketamine (125 pg/g) and xylazine (12.5 pg/g) and secured on a heating pad via taping of the paw tips.
  • additional IV anesthesia with pentobarbital (5 pg/g) was administered via the internal jugular catheter.
  • RNA Small interfering RNA
  • HAVECs Primary human umbilical vein endothelial cells
  • TNF-a-treated cells providing the exclusive source of TF and membrane surface, were then assayed for their ability to support factor Vila catalyzed conversion of factor X to factor Xa (FIG. 1A-D).
  • membrane PS composition is a critical determinant of factor X activation by the TF-VIIa complex (Bach and Rifkin, Proc Natl Acad Sci USA 87:6995-6999 (1990), Nemerson, J Clin Invest 47:72- 80 (1968), Ruf et al., J Biol Chem 1991;266:2158-2166 (1991), Wolberg et al., Blood Coagul Fibrinolysis 10:201-210 (1999)), our approach had the ability to identify regulators of anionic phospholipid membrane asymmetry that play a role in coagulation.
  • TMEM16E (AN05) was identified as the most significant positive regulator of factor Vila- catalyzed procoagulant activity in ECs (FIG. 1A).
  • TMEM16E is the closest paralog of the canonical Ca 2+ -activated PLS,TMEM16F (AN06) (Whitlock and Hartzell, Annu Rev Physiol 79: 119-143 (2017)), and TMEM16E has been shown to have PLS activity (Whitlock et al., J Gen Physiol 2018;150: 1498-1509 (2016), Gyobu et al., Mol Cell Biol 36:645-659 (2016)).
  • TMEM16F was also identified as a positive regulator of EC procoagulant activity (FIG. 1A).
  • TMEM16E and TMEM16F as PLSs that may promote coagulation on ECs.
  • TMEM16E and TMEM16F are required for procoagulant activity in ECs
  • TMEM16E and TMEM16F were tested.
  • Primary human ECs from various tissues express both TMEM16E and TMEM16F in a manner independent of TNF-a stimulation (FIG. 7A-C).
  • Two distinct siRNAs were confirmed to antagonize expression of TMEM16E or TMEM16F in ECs (FIG. 8A-E).
  • TMEM16E or TMEM16F were silenced in primary HUVECs and stimulated these cells with TNF-a to induce expression of TF.
  • Cells were then tested for their ability to support factor Vila catalyzed factor Xa generation assay in a one-stage factor Xa generation assay.
  • TMEM16E or TMEM16F Silencing of either TMEM16E or TMEM16F resulted in approximately 50% reduction of TF- dependent factor Xa generation compared to non-targeting control siRNA (FIG. IB and FIG. 1C). This reduction is comparable to that observed in cells treated with the PS-binding protein lactadherin (FIG. IB and FIG. 1C). Silencing of TMEM16E or TMEM16F also inhibited TF- dependent factor Xa generation that was augmented by addition of ionophore A23187 (FIG.
  • Ea.hy926 cell line stably expressing TF (Higgins et al., J Clin Invest 128:1471- 1484 (2016)) was used.
  • TMEM16E and TMEM16F were required for Ca 2+ ionophore-induced augmentation of TF-dependent factor X activation (FIG. 9).
  • ECs also required TMEM16E and TMEM16F to support plasma-derived thrombin generation following TNF-a stimulation (FIG. IE and FIG. IF).
  • EC-mediated thrombin generation was completely abolished by lactadherin (FIG. IF and FIG. 1G).
  • TMEM16E and TMEM16F function as Ca 2+ -activated PLSs, disrupting membrane phospholipid asymmetry by allowing PS and other anionic phospholipids to move down their concentration gradient from the inner to the outer membrane leaflet (Bevers and Williamson, Physiol Rev 96:605-645 (2016); Whitlock et al., J Gen Physiol 2018;150: 1498-1509 (2016); Yu et al., Elife 4: 1-23 (2015)).
  • TMEM16 proteins can also function as ion channels and have been implicated in regulating intracellular Ca 2+ flux in response to G-protein coupled receptor signaling (Yang et al., Cell 151 : 111-122 (2012); Yu et al., Elife 4: 1-23 (2015); Cabrita et al., FASEB J 31 :2123 -2134 (2017)). Therefore, TMEM16E or TMEM16F could affect PS externalization indirectly through regulation of Ca 2+ transients. To test this possibility, intracellular Ca 2+ was measured following stimulation with thrombin, which induces rapid Ca 2+ elevation in ECs. Silencing of TMEM16E or TMEM16F did not significantly affect intracellular Ca 2+ flux in ECs (FIG. 2E and FIG. 2F). These results suggest that TMEM16E and TMEM16F regulate EC procoagulant activity via PS externalization.
  • FIG. 11A-D PS externalization consistently extended proximal and distal to the site of laser ablation and in vessels of smaller diameter, often spread to the opposite wall (FIG. 3A-G and FIG. 11A-D).
  • Addition of the integrin a2bb3 (glycoprotein Ilb/IIIa) antagonist eptifibatide prevented platelet accumulation at the site of injury but had no effect on total PS extemalization at the site of injury (FIG. 3B-E).
  • Annexin V was employed at a dose of 0.025 pg/g body weight, which is significantly lower than annexin V doses shown to inhibit thrombosis in other models (Thiagarajan and Benedict, Circulation 96:2339-2347 (1997); Rbmisch et al., Thromb Res 61 :93-104 (1991)). Annexin V at this dose did, however, result in a mild reduction in fibrin formation, with no effect on platelet accumulation (FIG. 12A-E).
  • Use of other anionic phospholipid probes that bind PS such as lactadherin and pSIVA demonstrated an identical vessel wall pattern of PS exposure following laser injury (FIG. 3F and FIG. 3G).
  • mice lacking TMEM16E have reduced fibrin formation following vessel injury
  • TMEM16E is an important player in EC procoagulant activity
  • knockout of TMEM16E in mice could alter thrombosis.
  • Ano5 TMEM16E is highly expressed in skeletal muscle.
  • TMEM I 6E“ mice are overtly healthy but demonstrate defective muscle repair (Griffin et al., Hum Mol Genet 25: 1900-1911 (2016)).
  • blood coagulation in TMEM16E" ' mice has not been studied.
  • platelet and fibrin accumulation were monitored by intravital microscopy following laser injury of the cremasteric arteriole.
  • TMEM16E /_ mice demonstrated a small but significant decrease in fibrin formation (15.9%, median AUC 9.9 x 109 relative fluorescent units for TMEM16 + + and 8.3 x 109 for TMEM16E' ') and no difference in platelet accumulation compared to TMEM16E + + littermate controls (Figs. 4A-4D and FIG. 41).
  • animals were treated with eptifibatide to prevent platelet accumulation.
  • TMEM 16E mice Under eptifibatide treatment, TMEM 16E" mice demonstrated a more significant reduction in fibrin formation (43.5%, median AUC 1.53 x 10 10 relative fluorescent units [RFU] for TMEM16 +/+ and 0.66 x 10 10 for TMEM16E’ 7 ’) following laser injury compared to littermate controls (Figs. 4E-H and FIG. 4J). This suggested that platelet- mediated fibrin formation partially obscured TMEM16E-mediated thrombosis in animals not treated with eptifibatide. The diminished fibrin deposition in TMEM I 6E" mice could not be attributed to differences in baseline ex vivo coagulation parameters or platelet count (FIG. 13A- E).
  • TMEM16E was required for hemostasis
  • 8- to 10-week-old TMEM16E' ' and TMEM16E + + littermate control mice were tested in a tail clip bleeding assay.
  • TMEM16E" ' mice did not demonstrate excessive bleeding (Figs. 4K and 4L).
  • TMEM16 inhibitors reduce EC procoagulant activity
  • TMEM16 Pharmacologic inhibition of TMEM16 was tested to investigate if it could reduce EC procoagulant activity.
  • CaCCinh-AOl and benzbromarone are unrelated small molecules that have broad activity against TMEM16 channels including TMEM16E and TMEM16F (Zaitseva et al., Cell Host Microbe 22:99-110.
  • CaCCinh-AOl and benzbromarone each inhibited factor Xa generation in a dose-dependent manner with an IC50 of 2.0 pM and 3.2 pM, respectively (FIG. 5D).
  • IC50 2.0 pM and 3.2 pM, respectively.
  • CaCCinh-AOl and benzbromarone were tested to investigate whether reduced procoagulant activity is primarily via suppression of intracellular Ca 2+ elevation.
  • benzbromarone protects against thrombosis without increasing bleeding
  • benzbromarone was tested to investigate whether it demonstrates antithrombotic properties in vivo. Wild-type C57BL/J6 mice were treated with intraperitoneal injection of benzbromarone (5 pg/g body weight) one hour prior to assessing thrombus formation following laser injury of the cremasteric vasculature.
  • Benzbromarone reduced both platelet accumulation (55.1%, median AUC 6.28 x IO 10 RFU for vehicle and 2.82 x IO 10 for BBR) and fibrin formation (56.7%, median AUC 1.90 x 10 10 RFU for vehicle and 0.85 x 10 10 for BBR) following vessel injury (Figs. 6A-6D and 61). Mice were treated with eptifibatide to prevent platelet aggregation, and in the absence of platelet accumulation, benzbromarone still reduced fibrin formation (83.2%, median AUC 2.5 x 10 10 RFU for vehicle and 0.43 x 10 10 for BBR) following laser ablation (Figs. 6E-6H and 6 J). Benzbromarone did not result in excessive bleeding following tail amputation (Figs. 6K and 6L).
  • TMEM16E is identified as a novel positive regulator of PS externalization and procoagulant activity in ECs that may participate in thrombosis.
  • TMEM16 inhibitors decreased EC procoagulant activity and protected against thrombosis without increased bleeding complications.
  • monocytes (Del Conde et al., Arterioscler Thromb Vase Biol 25: 1065-1070 (2005)), erythrocytes (Wood et al., Blood 88:1873-1880 (1996); Bevers et al., Blood 79:380-388 (1992)), and ECs (Gao et al., PLoS One 2015;10: 1-16 (2015); Bombeli et al., Blood 89:2429-42 (1997);ffy et al., Blood 116:993-1001 (2010)) can also support coagulation in a PS-dependent manner.
  • the laser ablation model of vascular injury coupled with intravital microscopy has the ability to discern vessel wall- mediated versus platelet-mediated contributions to thrombosis (Higgins et al., J Clin Invest 128: 1471-1484 (2016); Jasuja et al., Blood 116:4665-4674 (2010); Vandendries et al., Proc Natl AcadSci 104:288-292 (2007); Kim et al., Blood 122: 1052-1061 (2013)).
  • PS Public Switching three different PS probes, it is observed that following vessel injury, accessible PS is primarily is localized on the vessel wall, not platelets, despite the formation of large platelet aggregates (FIG. 3A-G and FIG. 11A-D).
  • Annexin V sum intensity fluorescence did not correlate with the size of injury induced by laser ablation. This is in contrast to fibrin or platelet accumulation, which correlates well with injury size (FIG. 14A-C) (Grover et al., J Thromb Haemost 18:3078-3085 (2020)).
  • the lack of correlation between annexin V sum intensity and laser-induced injury size is likely due to in part to limitations of quantifying annexin V in a single Z-plane. In smaller vessels, PS extemalization often extends to the opposite vessel wall and results in a larger measured sum fluorescent intensity independent of injury size (FIG. 3A-G and FIG. 11A-D).
  • annexin V qualitative staining is very consistent, and the progression of PS externalization away from the site of injury occurs universally following laser ablation of all intensities.
  • Inflammatory stimuli such as lipopolysaccharide (Higgins et al., J Clin Invest 128: 1471-1484 (2016)) or TNF-a (this study) lead to concurrent expression of TF and externalization of PS, which together support procoagulant activity.
  • TF-dependent factor Xa generation is augmented by increasing PS externalization (FIG. 5B).
  • PS externalization in ECs similar to other cells, including platelets, appears to result from TMEM16 activation via sustained elevations in intracellular Ca 2+ (Bevers and Williamson, Physiol Rev 96:605-645 (2016)).
  • TMEM16E and TMEM16F couple Ca 2+ signaling to procoagulant activity in ECs.
  • TMEM16E and TMEM16F may affect Ca 2+ flux itself (Cabrita et al., FASEB J 31 :2123-2134 (2017)), whereas our data in ECs are similar to studies of platelets and lymphocytes from patients with Scott syndrome, where TMEM16F deficiency abolished PS externalization without inhibiting intracellular Ca 21 elevation (Munnix et al., Thromb Haemost 89:687-95 (2003); Kmit et al., Cell Death Dis 4: 1-8 (2013)).
  • PS exposure on the endothelium may therefore be a mechanism by which both apoptotic or non-apoptotic inflammatory stimuli contribute to thrombosis.
  • the caspase-activated PLS Xkr9 was also identified as a potential positive regulator of EC procoagulant activity (FIG. 1A). This finding may implicate other mechanisms of procoagulant PS externalization in ECs that may take on greater importance in distinct pathophysiologic states.
  • the TMEM16 family has 10 members, all of which act as Ca 2+ -activated ion channels, PLSs, or in certain cases, perhaps both (Whitlock and Hartzell, Anna Rev Physiol 79: 119-143 (2017)). The physiologic function of many family members is unknown.
  • TMEM16A and TMEM16B are bona fide Ca 2+ -activated chloride channels, whereas structural and biochemical studies establish a distinct “scrambling” domain of TMEM16F and its closest paralog TMEM16E that allows for transmembrane phospholipid exchange (Whitlock et al., J Gen Physiol 2018;150: 1498-1509 (2016); Gyobu et al., Mol Cell Biol 36:645-659 (2016); Yu et al., Elife 4:1- 23 (2015); Foltz et al., J Cell Biol 220:e202007059 (2021); Feng et al., Cell Rep 28:567-579 (2019)).
  • TMEM16F is ubiquitously expressed whereas TMEM16E exhibits tissue-restricted expression. High amounts of TMEM16E are found in skeletal muscle and most functional studies of TMEM16E have focused on muscle physiology. TMEM16E regulates muscle regeneration, myoblast fusion and myocyte membrane repair (Whitlock et al., J Gen Physiol 2018; 150: 1498- 1509 (2016); Griffin et al., Hum Mol Genet 25: 1900-1911 (2016); Sui et al., Cell Death Dis 9:609 (2018)).
  • TMEM16E membrane repair following injury
  • GMD2L limb girdle muscular dystrophy type 2L
  • GDD gnathodiaphyseal dysplasia
  • TMEM16E readily scrambles phospholipid, but the localization of TMEM16E within cells and importance of TMEM16E lipid scrambling per se, is unclear (Whitlock et al., Gen Physiol 2018;150:1498-1509 (2016); Gyobu et al., Mol Cell Biol 36:645-659 (2016)).
  • TMEM16E is predominantly expressed in intracellular compartments, such as the ER (Gyobu et al., Mol Cell Biol 36:645-659 (2016); Mizuta et al., Biochem Biophys Res Commun 357:126-132 (2007); Duran et al., Am J Physiol - Cell Physiol 302:482-493 (2012)), whereas others suggest it may also be located on the plasma membrane (Whitlock et al., J Gen Physiol 2018;150: 1498-1509 (2016); Di Zanni et al., Cell Mol Life Sci 75: 1657-1670 (2016)).
  • TMEM16E- null animals do not compensate by increasing expression of TMEM16F, for example (Foltz et al., JCellBiol 220:e202007059 (2021); Xu et al., Skelet Muscle 5: 1-14 (2015)). Dual silencing ofboth TMEM16E and TMEM16F did not result in additive reduction in procoagulant activity, likely because silencing of either TMEM16E or TMEM16F nearly completely inhibited PS extemalization (FIG. 1A-G and FIG. 2A-F).
  • TMEM16F-null mice demonstrated delayed occlusion in a ferric chloride carotid injury model of thrombosis and a mild bleeding diathesis (Yang et al., Cell 151 : 111-122 (2012)).
  • PITPa phosphatidylinositol transfer protein a
  • TMEM16 inhibition with two unrelated compounds reduced endothelial PS extemalization and procoagulant activity.
  • One such compound, the uricosuric agent benzbromarone has been used for decades to treat gout worldwide (Perez-Ruiz et al., Ann Rheum Dis 57:545-549 (1998); Heel et al., Drugs 14:349-66 (1977)).
  • TMEM16 inhibition could be further explored as an antithrombotic strategy, perhaps one targeting Ca 2+ -dependent PS extemalization during inflammation.
  • Benzbromarone is generally considered safe but was not approved by the FDA due to serious but exceedingly rare hepatotoxicity events estimated at 1 in 17,000 (Azevedo et al., Adv Rheumatol (London, England) 59:37 (2019)). Time-limited treatment with benzbromarone for inhibition of prothrombotic PS extern al izati on may have a more tolerable safety profile.
  • TMEM16 proteins as drivers of EC procoagulant activity.
  • Two PLSs are identified, TMEM16F, known to contribute to procoagulant activity in platelets, and TMEM16E, which has not previously been implicated in coagulation.
  • the vessel wall likely contributes procoagulant phospholipid to support thrombosis.
  • fibrin generation the terminal product in coagulation activation, is impaired in TMEM16E-deficient mice following vascular injury.
  • TMEM16 antagonism likely dampens prothrombotic endothelium and inhibits thrombosis without increasing bleeding complications. This study should foster future investigation into the significance of endothelial lipid scrambling in thrombotic disease and the role of TMEM16 inhibition as a viable antithrombotic target.

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Abstract

The present invention relates to methods of treating thrombotic disorders and hypercoagulation, including disorders related to COVID-19 and cancer. TMEM16 inhibitors are disclosed which inhibit development and progression of procoagulant endothelial cell dysfunction. Such inhibitors can be administered to a patient singly or in combination, and treating patients with a combination of complimentary TMEM16 inhibitors can result in beneficial, synergistic effects.

Description

COMPOSITIONS AND METHODS FOR REGULATING PROCOAGULANT ACTIVITY AND THROMBOSIS
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit under 35 U.S.C. §119(e) of U.S. Provisional Patent Application No. 63,387,083, filed December 12, 2022. The contents of these applications are incorporated herein by reference in their entireties for all purposes.
FIELD OF THE DISCLOSURE
[0002] The present disclosure relates to treatment of thrombosis and related disorders using
TMEM16 inhibitors alone or in combination.
GOVERNMENT SUPPORT CLAUSE
[0003] This invention was made with government support under Grant Nos. NHLBI DP5OD028129 and NHLBI K08HL161259 awarded by the NIH. The Government has certain rights in this invention.
SEQUENCE LISTING
[0004] The present disclosure contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on December 2, 2022, is named 148402-000400_Sequence_List_ST25.txt and is 3000 bites in size.
BACKGROUND
[0005] The present invention relates to treatment of thrombosis. TMEM16 proteins contribute to procoagulant activity in platelets, epithelial cells, and endothelial cells. Two phospholipid scrambiases are identified that contribute to processes that lead to thrombosis, including TMEM16F and TMEM16E. TMEM16E is newly identified in this invention as a contributor to thrombotic processes, and is involved in fibrin generation, the terminal product in coagulation activation. Targeting TMEM16 proteins with compounds that inhibit their activity reduces thrombosis without increasing bleeding complications. This invention highlights importance of modulation of TMEM16 inhibition for prevention and treatment of thrombotic diseases.
[0006] TMEM16 proteins are expressed in cells involved in physiological pathways that can lead to thrombosis, including both epithelial and endothelial cells. Endothelial cells (ECs) are constitutively an anticoagulant surface but switch to support coagulation following pathogenic stimuli. This switch promotes thrombotic cardiovascular disease. To generate thrombin at physiologic rates, coagulation proteins require a membrane surface containing anionic phospholipid, most notably phosphatidylserine (PS). PS can be rapidly externalized to the outer cell membrane leaflet by phospholipid “scrambiases”, such as TMEM16F. How ECs externalize phospholipids to support coagulation is not understood. A focused genetic screen was employed to evaluate the contribution of transmembrane phospholipid transport on EC procoagulant activity. Two TMEM16 family members were identified, TMEM16F, and its closest paralog, TMEM16E, which were both required to support coagulation on the EC surface via PS extemalization. Applying an intravital laser-injury model of thrombosis, PS extemalization was concentrated at the vessel wall and not in platelets. TMEM16E-null animals demonstrated reduced vessel walldependent fibrin formation. The TMEM16 inhibitor benzbromarone, used clinically for the treatment of gout, inhibited PS extemalization and EC procoagulant activity and protected animals from thrombosis without increasing bleeding. These findings indicate the activated endothelial surface is a source of procoagulant phospholipid contributing to thrombin formation. TMEM16 phospholipid scrambiases may be a valuable therapeutic target for thrombotic cardiovascular disease.
[0007] Thrombotic disorders such as myocardial infarction, stroke, and venous thromboembolism are leading causes of mortality worldwide (Wendelboe and Raskob, Circ Res 118: 1340-7 (2016)). Blood coagulation, and therefore thrombosis, depends at key stages on a membrane surface containing anionic phospholipid, most commonly phosphatidyl serine (PS) (Zwaal et al., Acta - Rev Biomembr 1376:433-453 (1998)). PS promotes the recruitment and activation of factor X by the tissue factor (TF)-factor Vila complex (Bach and Rifkin, Proc Natl Acad Set USA 87:6995-6999 (1990); Nemerson, J Clin Invest 47:72-80 (1968); Krishnaswamy et al., J Biol Chem 267:26110-26120 (1992)) and assembly and activity of the factor Xa-Va- prothrombin coagulation enzyme complex (Krishnaswamy et al., J Biol Chem 263:3823-3834 (1988); Rosing et al., J Biol Chem 255:274-283 (1980)), the initiating and ultimate steps in thrombin formation, respectively. Coagulation factor interaction with PS accelerates enzyme kinetics by at least 3 orders of magnitude to physiologic rates (Rosing et al., J Biol Chem 255:274- 283 (1980); Miletich et al., Proc Natl Acad Sci USA 74:4033-4036 (1977)). PS constitutes approximately 10-15% of plasma membrane phospholipid (Leventis and Grinstein, Amu Rev Biophys 39:407-427 (2010)), but under basal conditions is sequestered on the inner membrane leaflet and therefore inaccessible to the extracellular environment. A sustained rise in intracellular calcium (Ca2+) triggers PS extemalization to the outside of the cell (Balasubramanian et al., J Biol Chem 282: 18357-18364 (2007); Bevers et al., Biochim Biophys Acta 736:57-66 (1983)) by Ca2+- activated phospholipid scrambiases (PLSs), transmembrane channels that allow PS to move down its concentration gradient to the outer plasma membrane leaflet (Bevers and Williamson, Physiol Rev 96:605-645 (2016)). PS-binding proteins like annexin V and lactadherin prevent enzyme complex assembly and inhibit coagulation in vitro (Andree et al., J Biol Chem 267: 17907-17912 (1992); Ravanat et al., Biochem J 282:7-13 (1992); Shi et al., J. Thromb Haemost 6: 1167-1174 (2008)), and decrease thrombosis in vivo (Shi et al., J. Thromb Haemost 6: 1167-1174 (2008); Thiagarajan and Benedict, Circulation 96:2339-2347 (1997); Rbmisch et al., Thromb Res 61 :93- 104 (1991)). These findings suggest that PS exposure is an integral step in thrombosis and that targeting PS may be a viable antithrombotic strategy.
[0008] Following agonist stimulation in vitro, activated platelets readily externalize PS and support thrombin generation (Miletich et al., Proc Natl Acad Sci USA 74:4033-4036 (1977); Bevers et al., Biochim Biophys Acta 736:57-66 (1983)). The transition of platelets into a procoagulant form has provided an accessible means of studying cell-based PS exposure in coagulation. It has therefore been suggested that activated platelets are the major source of procoagulant PS in vivo and thus the major site of coagulation enzyme complex assembly for hemostasis and thrombosis (Heemskerk et al., Thromb Haemost 88: 186-193 (2002)). The identification of TMEM16F as a PLS required for PS extemalization in platelets (Suzuki et al., Nature 468:834-840 (2010); Yang et al., Cell 151 : 111-122 (2012)) and the finding that mutations in TMEM16F underlie the mild-to-moderate bleeding disorder Scott syndrome (Suzuki et al., Nature 468:834-840 (2010); Castoldi et al., Blood 117:4399-400 (2011)) launched investigation of TMEM16F in platelet function. Platelets require TMEM16F to externalize PS in response to stimuli that raise intracellular Ca2+ (Fujii et al., Proc Natl Acad Sci USA 112:12800-12805 (2015)). Loss of phospholipid scrambling in platelets protects from thrombosis or impairs hemostasis in some studies but not in others (Fujii et al., Proc Natl Acad Sci USA 1 12: 12800-12805 (2015); Baig et al., Arterioscler Thromb Vase Biol 36:2152-2157 (2016); Zhao et al., Nat Commun 8: 1-11 (2017); Mattheij et al., FASEB J 30:727-737 (2016)). It is therefore likely that sources of PS beyond the platelet and additional proteins beyond TMEM16F regulate procoagulant phospholipid extemalization.
[0009] Endothelial cells (ECs) form a constitutive anticoagulant surface under basal conditions to maintain blood flow. Loss of this anticoagulant property is considered a hallmark of cardiovascular disease leading to thrombosis. However, the relative contribution of activated endothelium to blood clotting in vivo is not well understood. Inflammatory stimuli such as hypoxia, cytokines, and lipopolysaccharide induce PS extemalization on ECs (Ran et al., Cancer Res 62:6132-40 (2002); Gao et al., PLoS One 2015;10: 1-16 (2015); Higgins et al., J Clin Invest 128: 1471-1484 (2018)), but the physiologic significance of EC phospholipid scrambling and the proteins that regulate it have not been characterized. Inflammatory stimuli also induce TF expression in ECs. The majority of cell surface-expressed TF binds factor Vll/VIIa but exists in a deactivated or “cryptic” state (REF), and PS extemalization is at least partly responsible for enhancing the factor Vila cofactor activity of TF (Ruf et al., J Biol Chem 1991;266:2158-2166 (1991); Wolberg et al., Blood Coagitl Fibrinolysis 10:201-210 (1999)). Therefore, identification of regulators of PS extemalization will aid our understanding of TF activation and inflammatory thrombosis.
[0010] A targeted screen was performed of genes encoding proteins predicted to regulate transmembrane phospholipid transport for their contribution to TF-dependent factor Xa generation in ECs. This approach identified two TMEM16 family members, TMEM16F, and its closest paralog, TMEM16E (Whitlock and Hartzell, Annu Rev Physiol 79: 119-143 (2017)), that support EC procoagulant activity. The TMEM16 family are all transmembrane proteins, activated by Ca2+, and function as ion channels, PLSs, or in some cases both (Whitlock and Hartzell, Anna Rev Physiol 79:119-143 (2017)). TMEM16E is highly expressed in skeletal muscle, where it regulates muscle regeneration and repair (Whitlock et al., J Gen Physiol 2018;150: 1498-1509 (2018); Griffin et al., Hum Mol Genet 25: 1900-1911 (2016)), but is not expressed in platelets (Fujii et al., Proc Natl Acad Sci USA 112:12800-12805 (2015); Rowley et al., Blood 118:el01-el 11 (2011)) and has no previously known role in hemostasis or thrombosis. Our studies demonstrate that both TMEM16E and TMEM16F controlled PS extemalization in ECs and are necessary to support coagulation on the endothelial surface. Using a well -validated and widely reproduced intravital laser-ablation model of thrombosis (Falati et al., Nat Med 8: 1175-1180 (2002)), it was observed that the majority of PS at the site of thrombus formation in vivo originates from the vessel wall, independent of platelets, and mice lacking TMEM16E demonstrate reduced fibrin formation following vessel injury. Pharmacologic inhibition of TMEM16 proteins with benzbromarone decreases PS extemalization and procoagulant activity of ECs, and protects mice from thrombosis without increasing bleeding time. These results suggest that EC -derived PS contributes to thrombus formation in vivo and that small-molecule inhibition of TMEM16 may be a novel antithrombotic strategy.
BRIEF DESCRIPTION OF THE DRAWINGS
[0011] FIG. 1A-G shows that TMEM16E and TMEM16F are required for TF activity and thrombin generation on ECs. FIG.1A shows a heat map illustrating the relative positive or negative regulation of factor Vila catalyzed factor X activation following silencing of the indicated genes in HUVECs. Pools of four distinct siRNA were directed at the indicated target and tested in triplicate. Each box represents an independent experimental plate with scale bar depicting percentage of factor Xa generated compared to cells transfected with untargeted control siRNA. Lighter color indicates lower percentage factor Xa generation compared to control. Figs. 1B-G. HUVECs were transfected with individual siRNAs for 72 h and assayed for their ability to support factor Vila catalyzed factor Xa generation (Figs. 1B-D) or thrombin generation in plasma-treated ECs (Figs. 1E-G). Cells were stimulated with TNF-a (10 ng/mL) for 3.5 h (FIG. IB, FIG. 1C, FIG. IE, and FIG. IF), TNF-a for 3.5 h plus Ca2+ ionophore A23187 (6 pM) for 20 min (FIG. ID) or A23187 alone for 20 min (FIG. 1G). Representative experiments are depicted as mean absorbance for factor Xa generation (FIG. IB) or the first derivative of arbitrary fluorescent units for thrombin generation (FIG. IE), as a function of time. 16E, 16F and TF denote siRNA targeting TMEM16E, TMEM16F, and tissue factor, respectively. #1 and #2 denote distinct siRNA sequences. Lact denotes lactadherin (100 nM), added in addition to control siRNA, n = 3-5 independent experiments. Error bars indicate mean ± SEM (FIG. IB and FIG. IE) or mean ± SD (FIG. 1C, FIG. ID, FIG. IF, and FIG. 1G), ANOVA with Tukey’s posttest, *p< 0.05, ”p<0.01, ""pO.OOOl . [0012] FIG. 2A-F shows that TMEM16E and TMEM16F are required for phosphatidyl serine (PS) external! zati on on ECs. HUVECs were transfected with indicated siRNAs for 72 h, stimulated with TNF-a (10 ng/mL) for 18 h (FIG. 2A) or Ca2+ ionophore A23187 (6 pM) for 20 min (FIG. 2B), and stained with annexin V (green) to detect PS externalization and Zombie Red (red) to detect cell death. Total annexin V fluorescence for each image was normalized to number of nuclei (blue) and dead (Zombie Red-positive, red) cells. FIG. 2C. PS externalization following treatment with ionophore A23187 (6 pM) was detected using annexin V by flow cytometry. Histograms are generated from gating on live (DAPI-negative) cells only. FIG. 2D. HUVECs were transfected with indicated siRNAs for 72h, stimulated with TNF-a (10 ng/mL) and stained for TF (green). Mean fluorescent intensity (MFI) was normalized to background for each image. Representative images are shown for each experiment. FIG. 2E and FIG. 2F. HUVECs were transfected with indicated siRNAs for 72 h, and intracellular Ca2+ flux was measured with the Ca2+-sensitive dye Calbryte 520 AM following stimulation with thrombin (1 U/mL). Silencing of the store-operated Ca21 regulator STIM1 served as a positive control. Time course of Calbryte 520 fluorescence after thrombin stimulation, normalized to background fluorescence (FIG. 2E) and area under the curve (AUC) values (FIG. 2F), normalized to cells treated with control siRNA. 16E, 16F, and TF denote siRNA targeting TMEM16E, TMEM16F, and tissue factor, respectively. #1 and #2 denote different siRNA sequences. Scale bar is 50 pm in FIG. 2A and FIG. 2B and 100 pm in FIG. 2D. n = 3-6 independent experiments. Error bars indicate mean ± SD (FIG. 2A-D and FIG. 2F) or mean ± SEM in (FIG. 2E). ANOVA with Tukey’s posttest *p<0.05, "p<0.01, ***p<0.001, ****p<0.0001.
[0013] FIG. 3A-G shows that PS externalization visualized via intravital microscopy occurs on the vessel wall and is unaffected by platelet inhibition. Thrombus formation was monitored for 180 seconds in wild-type mice following laser injury of the cremasteric arteriole in the presence or absence of the platelet aggregation inhibitor eptifibatide (10 pg/g body weight). FIG. 3A. Representative images at indicated time points of the PS probe annexin V (red, Alexa fluor-647), platelets (anti-CD42b antibody, blue, Dylight 405), and fibrin (anti-fibrin antibody, green, Dylight 488). Note annexin V positivity on the vessel wall and in the absence of platelet aggregation. Kinetics and magnitude of median integrated relative fluorescent units (RFU) for platelet accumulation (FIG. 3B) and PS externalization (FIG. 3D) are shown following laser injury. Area under the curve (AUC) for fluorescent intensity was determined for platelets (FIG. 3C) and annexin V (FIG. 3E). Line represents the median AUC for individual thrombi (Vehicle n = 38, Eptifibatide n = 38) analyzed by Mann-Whitney, ****p<0.0001. A vessel-wall pattern for PS extemalization is also observed using alternative PS probes pSIVA (FIG. 3F, red pseudocolor) and lactadherin-FITC (FIG. 3G, red pseudocolor), 180 sec following laser injury. In both FIG. 3F and FIG. 3G, platelets are labeled blue. Representative images are shown from 10 individual thrombi. Arrowheads denote extent of vessel-wall injury and “X” indicates site of laser ablation. Arrows indicate extension of PS extemalization to the opposite vessel wall. Scale bar is 25 pm.
[0014] FIG. 4A-L shows that TMEM16E supports vessel wall fibrin formation. Thrombus formation following laser injury of the cremasteric arteriole was monitored for 180 sec in TMEM16E /_ (^4no5'/_) or TMEM16E+/+ (Ano5 ) littermate controls in the presence of vehicle (FIG. 4A-D) or eptifibatide 10 pg/g body weight (FIG. 4E-H). Platelet and fibrin accumulation were monitored by anti-CD42b and anti-fibrin antibody conjugated to Dylight 405 and 488, respectively. Kinetics and magnitude of median integrated relative fluorescent units (RFU) for platelet (FIG. 4A and FIG. 4E) and fibrin (FIG. 4C and FIG. 4G) accumulation are shown following laser injury. Area under the curve (AUC) for fluorescent intensity was determined for platelets (FIG. 4B and FIG. 4F), and fibrin (FIG. 4D and FIG. 4H) for each thrombus. Line represents the median AUC for individual thrombi (Vehicle 16E+ + n = 52, 16E“ n = 54; Eptifibatide 16E+/+ n= 45 16E" ' n = 39) analyzed by Mann-Whitney, *p<0.05, **p<0.01. Injury sizes associated with the thrombi analyzed above under vehicle (FIG. 41) and eptifibatide (FIG. 4J), analyzed by Student’s t test, *p<0.05. Time to cessation of bleeding (FIG. 4K) and total hemoglobin loss (FIG. 4L) were measured following tail clipping (16E+ + n = 10, 16E" n = 10), analyzed by Mann-Whitney.
[0015] FIG. 5A-F shows thatTMEM16 antagonists reduce EC procoagulant activity by inhibiting PS extemalization. FIG. 5A. HUVECs were treated with Ca2+ ionophore A23187 at indicated concentrations for 20 min and PS extemalization was determined by annexin V binding, measured by flow cytometry. Histograms are generated from gating on live (D API-negative) cells only. FIG. 5B. HUVECs were treated with both TNF-a (10 ng/mL) for 3.5 h and A23187 for 20 min at indicated concentrations and analyzed for factor Vila catalyzed conversion of factor X to factor Xa. Anti-TF antibody and lactadherin (Lact,100 pM) were used to block TF and PS, respectively. FIG. 5C. HUVECs were treated with A23187 (6 pM) in the presence of TMEM16 inhibitors CaCCinh-AOl (A01) and benzbromarone (BBR) (10 pM) and analyzed for PS extemalization as in FIG. 5A. FIG. 5D. TMEM16 antagonists were assayed for their ability to inhibit factor Xa generation on HUVECs stimulated with TNF-a (10 ng/mL, 3.5 h) followed by A23187 (6 pM, 20 min). FIG. 5E and FIG. 5F. HUVECs were treated with TMEM16 inhibitors A01 or BBR, or the phospholipase C inhibitor U73122, and intracellular Ca2 transients were measured with Calbryte 520 AM following stimulation with thrombin (1 U/mL). Time course of Calbryte 520 fluorescence after thrombin stimulation, normalized to background fluorescence (FIG. 5E) and area under the curve (AUC) values (FIG. 5F), normalized to cells treated with DMSO vehicle control, n = 3-6 independent experiments. Error bars indicate mean ± SD (FIG. 5A-D and FIG. 5F) or mean ± SEM in (FIG. 5E). ANO VA with Tukey’s posttest, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.
[0016] FIG. 6A-L shows that benzbromarone inhibits thrombosis without increasing bleeding. Thrombus formation following laser injury of the cremasteric arteriole was monitored for 180 sec in mice treated with benzbromarone (BBR 5 pg/g) or vehicle (FIG. 6A-D and FIG. 61) and BBR or vehicle in the presence of eptifibatide (10 pg/g body weight) (FIG. 6E-H and FIG. 6J). Platelet and fibrin accumulation were monitored by anti-CD42b and anti-fibrin antibody conjugated to Dylight 405 and 488, respectively. Kinetics and magnitude of median integrated relative fluorescent units (RFU) for platelet (FIG. 6A and FIG. 6E) and fibrin (FIG. 6C and FIG. 6G) accumulation are shown following laser injury. Area under the curve (AUC) for fluorescent intensity was determined for platelets (FIG. 6B and FIG. 6F), fibrin (FIG. 6D and FIG. 6H) for each thrombus. Line represents the median AUC for individual thrombi (Vehicle n = 46, BBR n = 54; eptifibatide + vehicle n= 37 eptifibatide + BBR n = 40) analyzed by Mann-Whitney, *p<0.05, **p<0.01, ***p<0.001. FIG. 61 and FIG. 6J. Injury sizes associated with the thrombi analyzed above, analyzed by Student’s t test. Time to cessation of bleeding (FIG. 6K) and total hemoglobin loss (FIG. 6L) were analyzed following tail clipping in mice treated with BBR (Vehicle n = 12, BBR n = 10), analyzed by Mann- Whitney.
[0017] FIG. 7A-C shows expression of TMEM16E and TMEM16F in primary endothelial cells. Messenger RNA from HUVECs (FIG. 7A), coronary artery endothelial cells (CAEC, FIG. 7B) and lung microvascular endothelial cells (HMVEC-L, FIG. 7C) following stimulation with TNF-a (10 ng/mL) was analyzed by qPCR. Relative mRNA level is compared to that of b- actin. n = 3 independent experiments, Error bars indicate mean ± SD. [0018] FIG. 8A-E shows validation of siRNA targeting TMEM16E and TMEM16F. TMEM16E protein could be readily detected when overexpressed in HUVECs via lentiviral transduction. FIG. 8A. HUVECs stably expressing TMEM16E containing a C-terminal V5 tag were transfected with indicated siRNA targeting TMEM16E or untargeted control siRNA for 72 h. TMEM16E protein was determined by SDS-PAGE and immunoblotting with antibodies against TMEM16E, V5, and actin (loading control). TMEM16E is detected as a band running just above 100 kd. FIG. 8B. Primary HUVECs were transfected with siRNA targeting TMEM16E for 72 h before determining TMEM16E mRNA level by quantitative PCR. FIG. 8C. HUVECs were transfected with siRNA targeting TMEM16F for 72 h. TMEM16F protein was determined by SDS- PAGE and immunoblotting with anti-TMEM16F antibody. FIG. 8D. Primary HUVECs or HUVECs stably expressing TMEM16E-V5 were transfected with indicated siRNA for 72 h prior to determination of TMEM16E and TMEM16F protein by SDS-PAGE and immunoblotting with anti-TMEM16F and anti-V5 antibody. FIG. 8E. HUVECs were transfected with indicated siRNA for 72 h and cell viability was determined by XTT assay. #1 and #2 denote independent siRNA sequences. TF denotes siRNA targeting tissue factor.
[0019] FIG. 9 shows that TMEM16E and TMEM16F regulate tissue factor procoagulant activity. An Ea.hy926 cell line stably expressing tissue factor was transfected with siRNAs targeting TMEM16E, TMEM16F, or TF for 72 h. Cells were treated with Ca2+ ionophore A23187 (6 pM) for 20 min and assayed for their ability to support factor Vila catalyzed conversion of factor X to factor Xa. 16E, 16F and TF denote siRNA targeting TMEM16E, TMEM16F, and tissue factor, respectively. #1 and #2 denote independent siRNA sequences. Error bars indicate mean ± SD, ANOVA with Tukey’s posttest, ****p<0.0001.
[0020] FIG. 10A-C shows that TMEM16E and TMEM16F are required for PS extemalization on ECs. HUVECs were transfected with indicated siRNAs for 72 h, stimulated with TNF-a (10 ng/mL) for 18 h (FIG. 10A) or calcium ionophore A23187 (6 pM) for 20 min (FIG. 10B), and stained with annexin V to detect PS extemalization and Zombie Red to detect cell death. Each dot represents the total fluorescent area of Zombie Red per image normalized to the number of nuclei present. Note no increase in cell death (Zombie Red positivity) in cells treated with TNF-a or A23187 compared to control. FIG. 10C. Representative flow cytometric analysis of PS exposure (annexin V, x-axis) and cell death (DAPI, y-axis). Numbers refer to the percentage of total cells in each quadrant. QI represents dead, PS-negative population, Q2 represents dead, PS-positive population, Q3 represents live, PS-positive population, Q4 represents live, PSnegative population.
[0021] FIG. 11A-C shows phosphatidylserine externalization during thrombus formation following laser injury. Thrombus formation was monitored for 180 seconds in wild-type mice following laser injury of the cremasteric arteriole (FIG. 11A) and additionally in the presence of the platelet aggregation inhibitor eptifibatide (FIG. 11B, 10 pg/g of body weight). Representative images at indicated time points of the PS probe annexin V (red, Alexa Fluor 647), platelets (anti- CD42b antibody, blue, Dylight 405), and fibrin (anti-fibrin antibody, green, Dylight 488). Note annexin V positivity on the vessel wall in the absence of platelet aggregation. Arrowheads denote extension of vessel-wall injury and “X” indicates site of laser ablation. Arrows indicate extension of annexin V binding to the opposite vessel wall. To better visualize annexin V binding, platelet fluorescence is omitted from the bottom images in (FIG. 11A). Asterisk (*) indicates the platelet aggregate. FIG. 11C. Annexin V staining following laser ablation. Annexin V is often observed wrapping around the vessel to the opposite wall. Dotted yellow lines indicate the vessel wall boundaries. FIG. 11D. 3-dimensional renderings of Z-stack images of annexin V binding following laser injury from the image in FIG. 11C. Scale bar is 25 pm unless otherwise indicated. [0022] FIG. 12A-E shows that annexin V inhibits thrombosis. Thrombus formation was monitored for 180 seconds in wild-type mice following laser injury of the cremasteric arteriole in the presence or absence of annexin V (0.025 pg/g of body weight). Platelet and fibrin accumulation were monitored by anti-CD42b and anti-fibrin antibody conjugated to Dylight 647 and 488, respectively. Kinetics and magnitude of median integrated relative fluorescent units (RFU) for platelet (FIG. 12A) and fibrin (FIG. 12C) accumulation are shown following laser injury. The area under the curve (AUC) for fluorescent intensity was determined for platelets (FIG. 12B), fibrin (FIG. 12D) for each thrombus, analyzed by Mann-Whitney. FIG. 12E. Injury sizes associated with the thrombi analyzed above, analyzed by Student’s t test.
[0023] FIG. 13A-E shows blood and coagulation parameters in TMEM16E" ' mice. Blood from TMEM16E’ ’ (AHO5' ~) and TMEM16+/+ (Ano5 ) littermate controls was assessed for white blood cells (WBC, FIG. 13A), hemoglobin (Hg, FIG. 13B), and platelet counts (FIG. 13C). Plasma was assessed for prothrombin time (PT, FIG. 13D) and activated partial thromboplastin time (aPTT, FIG. 13E). Error bars indicate mean ± SEM, n = 5-7 animals per genotype. [0024] FIG. 14A -C shows correlation of annexin V binding with vessel wall injury size. Injury size following laser ablation is plotting against the area under the curve (AUC) for platelets (FIG. 14A), fibrin (FIG. 14B), and annexin V (FIG. 14C) demonstrate a strong Spearman correlation for platelets and fibrin (p < 0.0001) but not for annexin V.
SUMMARY
[0025] Embodiments provided herein include a method of treating a thrombotic disorder, including administering a therapeutically effective amount of one or more TMEM16 inhibitors to a patient in need thereof. In certain embodiments, the TMEM16 is TMEM16E, TMEM16F, or any combination thereof. In some embodiments, the one or more TMEM16 inhibitors is benzbromarone, isoquercetin, quercetin aglycone, quercetin derivatives, quercetin-3-O-glucoside (also known as isoquercetin), quercetin-5-O-glucoside, quercetin-7-O-glucoside, quercetin-9-O- glucoside quercetin-3’-O-glucoside, quercetin-4’-O-glucoside, quercetin-3-O-rutinoside (also known as rutin), quercetin-3-O-[a-rhamnosyl- (l->2)-a-rhamnosyl -(l->6)]-I3-glucoside, quercetin-3-O-galactoside, quercetin-7-O-galactoside, quercetin-3-O-rhamnoside, quercetin-7-O- galactoside, quercetin-glycoside, 7-hydroxyflavone, rutin, any constituent of rutin or isoquercetin, metabolite of rutin or isoquercetin or quercetin, a sulphated, glucuronidated or methylated form of rutin or quercetin, ivermectin, crofelemer, nitazoxanide, hexachlorophene, dichlorophen, nitazoxanide, trifluoperazine, or a combination thereof. In some embodiments, the one or more TMEM16 inhibitors includes benzbromarone. In certain embodiments, the one or more TMEM16 inhibitors further include isoquercetin.
[0026] In certain embodiments, the one or more TMEM16 inhibitors include benzbromarone and isoquercetin, and administering benzbromarone and isoquercetin results in a synergistic antithrombotic effect.
[0027] Additional embodiments of the method provided herein include also administering zafirlukast. In certain embodiments of the method described herein, administering one or more TMEM16 inhibitors, and additionally administering zafirlukast, results in a synergistic antithrombotic effect.
[0028] In some embodiments of the method provided herein, a therapeutically effective amount of benzbromarone is from about 50 mg to about 800 mg per day. In some embodiments of the method described herein, a therapeutically effective plasma concentration of benzbromarone is from about 1 to about 30 micromolar. In certain embodiments, a therapeutically effective amount of benzbromarone is about 5 pg/g. In some embodiments, the therapeutically effective amount of benzbromarone is administered between about every 12 to about every 24 hours. In some embodiments, the therapeutically effective amount of benzbromarone is administered about every 24 hours.
[0029] In further embodiments of the method provided herein, the thrombotic disorder is a thrombotic disease, hypercoagulation, blood coagulation, TMEM16-induced coagulation, coronary artery disease, cardiovascular disease, procoagulant endothelial cell dysfunction, procoagulant pulmonary epithelial cell dysfunction, aberrant procoagulant activity, pathological phosphoserine extemalization, dysregulated blood coagulation, antiphospholipid antibody syndrome, SARS-CoV-2 pneumonia, thrombosis during severe SARS-CoV-2 infection, post-acute sequelae of SARS CoV-2 infection, cancer-associated thrombosis, refractory thrombosis, myocardial infarction, stroke, venous thromboembolism, disseminated intravascular coagulation (DIC), sepsis, paroxysmal nocturnal hemoglobinuria, antiphospholipid antibody syndrome, myeloproliferative disorders, inherited red cell disorders associated with thrombosis, sickle cell disease, hereditary xerocytosis, acute coronary syndrome, atrial fibrillation, or any combination thereof.
[0030] In some embodiments, the method relates to endothelial cells, epithelial cells, cancer cells, or other cells that can be involved in thrombotic diseases.
[0031] In certain embodiments, administering the therapeutically effective amount of one or more TMEM16 inhibitors does not increase bleeding complications, bleeding time, bleeding risk, or any combination thereof.
[0032] In some embodiments of the method provided herein, administering the therapeutically effective amount of one or more TMEM16 inhibitors results in a reduction in at least one indicator of thrombotic disease, results in at least one indicator of thrombotic disease remaining within an acceptable range, or a combination thereof. In further embodiments, the at least one indicator of thrombotic disease is D-dimer, thrombin antithrombin complex, prothrombin activation peptide Fl+2, procoagulant extracellular vesicle TF cofactor activity, factor Xa generation, thrombin generation, or plasma endogenous thrombin potential. In certain embodiments, administering the therapeutically effective amount of one or more TMEM16 inhibitors results in concentrations of D-dimer in the patient remaining below about 500 ng/mL. [0033] In certain embodiments of the method provided herein, the one or more TMEM16 inhibitors is administered in combination with a blood-thinning medication, chemotherapy, or a drug indicated for the treatment of heart disease. In some embodiments, the one or more TMEM16 inhibitors is administered in combination with warfarin, heparin, low molecular weight heparins, ultra-low molecular weight heparins, direct oral anticoagulants targeting thrombin, factor Xa, factor Xia, parenteral direct thrombin inhibitors, argatroban, aspirin, P2Y12 inhibitors, clopidogrel, or a combination thereof.
[0034] In certain embodiments, the thrombotic disorder is associated with COVID- 19.
[0035] In further embodiments of the method described herein, the patient has cancer. In certain embodiments, the cancer is multiple myeloma, hematologic cancer, adenocarcinoma, cancer of the pancreas, stomach, ovaries, prostate, colon, lung, brain, breast, kidney, skin, cervix, or ear-nose-throat cancer.
[0036] Further embodiments of the method provided herein are directed to a method for treating hypercoagulation related to COVID-19, including administering a therapeutically effective amount of one or more TMEM16 inhibitors to a patient in need thereof. In certain embodiments, the COVID-19 is post-acute sequelae of SARS CoV-2 infection.
[0037] In some embodiments of the method provided herein, the TMEM16 is TMEM16E, TMEM16F, or any combination thereof. In certain embodiments, the one or more TMEM16 inhibitors is benzbromarone, isoquercetin, ivermectin, crofelemer, nitazoxanide, hexachlorophene, dichlorophen, nitazoxanide, trifluoperazine, or a combination thereof. In certain embodiments, the one or more TMEM16 inhibitors includes benzbromarone.
[0038] In certain embodiments of the method provided herein, the one or more TMEM16 inhibitors further includes isoquercetin. In some embodiments, administering the one or more TMEM16 inhibitors including benzbromarone and isoquercetin results in a synergistic antihypercoagulation effect.
[0039] In certain embodiments, a therapeutically effective amount of benzbromarone is from about 50 mg to about 800 mg per day. In some embodiments, a therapeutically effective plasma concentration of benzbromarone is from about 1 to about 30 micromolar. In some embodiments, a therapeutically effective amount of benzbromarone is about 5 pg/g. In some embodiments, the therapeutically effective amount of benzbromarone is administered between about every 12 to about every 24 hours. In certain embodiments, the therapeutically effective amount of benzbromarone is administered about every 24 hours.
[0040] In some embodiments of the method provided herein, the hypercoagulation related to COVID-19 is microcoagulation, microvascular thrombosis, immunothrombosis, TMEM16- induced coagulation, coronary artery disease, cardiovascular disease, procoagulant endothelial cell dysfunction, procoagulant pulmonary epithelial cell dysfunction, aberrant procoagulant activity, pathological phosphoserine externalization, dysregulated blood coagulation, antiphospholipid antibody syndrome, SARS-CoV-2 pneumonia, thrombosis during severe SARS-CoV-2 infection, refractory thrombosis, myocardial infarction, stroke, venous thromboembolism, disseminated intravascular coagulation (DIC), sepsis, paroxysmal nocturnal hemoglobinuria, antiphospholipid antibody syndrome, myeloproliferative disorders, acute coronary syndrome, atrial fibrillation, or any combination thereof.
[0041] Additional embodiments provide that administering the therapeutically effective amount of one or more TMEM16 inhibitors does not increase bleeding complications, bleeding time, bleeding risk, or any combination thereof.
[0042] In further embodiments, administering the therapeutically effective amount of one or more TMEM16 inhibitors results in a reduction in at least one indicator of hypercoagulation, results in at least one indicator of hypercoagulation remaining within an acceptable range, or a combination thereof. In some embodiments, the at least one indicator of hypercoagulation is D- dimer, thrombin antithrombin complex, prothrombin activation peptide Fl+2, procoagulant extracellular vesicle TF cofactor activity, factor Xa generation, thrombin generation, or plasma endogenous thrombin potential. In some embodiments, administering the therapeutically effective amount of one or more TMEM16 inhibitors results in concentrations of D-dimer in the patient remaining below about 500 ng/mL.
[0043] In some embodiments, the one or more TMEM16 inhibitors is administered in combination with oral antiviral drugs, intravenous antiviral drugs, monoclonal antibodies, bloodthinning medications, or a drug indicated for the treatment of heart disease. In further embodiments, the one or more TMEM 16 inhibitors is administered in combination with direct oral anticoagulants, dabigatran, rivaroxaban, apixaban, edoxaban, betrixaban, Paxlovid, molnupiravir, remdesivir, Bebtelovimab, Evusheld, warfarin, heparin, low molecular weight heparins, ultra-low molecular weight heparins, direct oral anticoagulants targeting thrombin, factor Xa, factor Xia, parenteral direct thrombin inhibitors, argatroban, aspirin, P2Y12 inhibitors, clopidogrel, or a combination thereof.
[0044] In additional embodiments, the method described herein also includes administering zafirlukast. In certain embodiments, administering the one or more TMEM16 inhibitors, including administering zafirlukast, results in a synergistic anti-hypercoagulation effect. [0045] Further embodiments of the method provided herein include a method for reducing cancer-associated thrombosis, including administering to a patient a therapeutically effective amount of one or more TMEM16 inhibitors. In certain embodiments, the TMEM16 is TMEM16E, TMEM16F, or any combination thereof. In some embodiments, the one or more TMEM16 inhibitors is benzbromarone, isoquercetin, ivermectin, crofelemer, nitazoxanide, hexachlorophene, dichlorophen, nitazoxanide, trifluoperazine, or a combination thereof. In certain embodiments, the one or more TMEM16 inhibitors includes benzbromarone.
[0046] In some embodiments, the one or more TMEM16 inhibitors further includes isoquercetin. In certain embodiments, administering the one or more TMEM16 inhibitors including benzbromarone and isoquercetin results in a synergistic antithrombotic effect.
[0047] In certain embodiments, the method described herein further includes administering zafirlukast. In certain embodiments, administering the one or more TMEM16 inhibitors with zafirlukast results in a synergistic antithrombotic effect.
[0048] In certain embodiments, a therapeutically effective amount of benzbromarone is from about 50 mg to about 800 mg per day. In some embodiments, a therapeutically effective plasma concentration of benzbromarone is from about 1 to about 30 micromolar. In some embodiments, a therapeutically effective amount of benzbromarone is about 5 pg/g. In some embodiments, the therapeutically effective amount of benzbromarone is administered between about every 12 to about every 24 hours. In certain embodiments, the therapeutically effective amount of benzbromarone is administered about every 24 hours.
[0049] In further embodiments, the patient has a thrombotic disease, cardiovascular disease, procoagulant endothelial cell dysfunction, procoagulant pulmonary epithelial cell dysfunction, aberrant procoagulant activity, pathological phosphoserine extemalization, dysregulated blood coagulation, antiphospholipid antibody syndrome, cancer-associated thrombosis, refractory thrombosis, myocardial infarction, stroke, venous thromboembolism, disseminated intravascular coagulation (DIC), sepsis, paroxysmal nocturnal hemoglobinuria, antiphospholipid antibody syndrome, myeloproliferative disorders, inherited red cell disorders associated with thrombosis, sickle cell disease, hereditary xerocytosis, acute coronary syndrome, atrial fibrillation, or any combination thereof.
[0050] In some embodiments of the method provided herein, administering the therapeutically effective amount of one or more TMEM16 inhibitors does not increase bleeding complications, bleeding time, bleeding risk, or any combination thereof.
[0051] In certain embodiments, administering the therapeutically effective amount of one or more TMEM16 inhibitors results in a reduction in at least one indicator of thrombotic disease, results in at least one indicator of thrombotic disease remaining within an acceptable range, or a combination thereof. In some embodiments, the at least one indicator of thrombotic disease is D- dimer, thrombin antithrombin complex, prothrombin activation peptide Fl+2, procoagulant extracellular vesicle TF cofactor activity, factor Xa generation, thrombin generation, or plasma endogenous thrombin potential. In certain embodiments, administering the therapeutically effective amount of one or more TMEM16 inhibitors results in concentrations of D-dimer in the patient remaining below about 500 ng/mL.
[0052] In some embodiments of the method provided herein, the one or more TMEM16 inhibitors is administered in combination with a blood-thinning medication, chemotherapy, or a drug indicated for the treatment of heart disease. In certain embodiments, the one or more TMEM16 inhibitors is administered in combination with warfarin, heparin, low molecular weight heparins, ultra-low molecular weight heparins, direct oral anticoagulants targeting thrombin, factor Xa, factor Xia, parenteral direct thrombin inhibitors, argatroban, aspirin, P2Y12 inhibitors, clopidogrel, or a combination thereof.
[0053] In certain embodiments, the cancer is multiple myeloma, hematologic cancer, adenocarcinoma, cancer of the pancreas, stomach, ovaries, prostate, colon, lung, brain, breast, kidney, skin, cervix, or ear-nose-throat cancer.
[0054] Further embodiments provided herein include a therapeutic composition suitable to treat a thrombotic disorder, including a therapeutically effective amount of one or more TMEM16 inhibitors, and pharmaceutically acceptable excipients.
[0055] In certain embodiments of the therapeutic composition provided herein, the TMEM16 is TMEM16E, TMEM16F, or any combination thereof. In some embodiments, the one or more TMEM16 inhibitors is benzbromarone, isoquercetin, ivermectin, crofelemer, nitazoxanide, hexachlorophene, dichlorophen, nitazoxanide, trifluoperazine, or a combination thereof. In some embodiments, the one or more TMEM16 inhibitors includes benzbromarone.
[0056] In certain embodiments, the one or more TMEM16 inhibitors further includes isoquercetin. In some embodiments, administering the one or more TMEM16 inhibitors including benzbromarone and isoquercetin results in a synergistic antithrombotic effect.
[0057] In certain embodiments, the therapeutic composition described herein further includes administering zafirlukast. In some embodiments, administering the one or more TMEM16 inhibitors, and further including administering zafirlukast, results in a synergistic antithrombotic effect.
[0058] In some embodiments of the method provided herein, a therapeutically effective amount of benzbromarone is from about 50 mg to about 800 mg per day. In some embodiments of the method described herein, a therapeutically effective plasma concentration of benzbromarone is from about 1 to about 30 micromolar. In certain embodiments, a therapeutically effective amount of benzbromarone is about 5 pg/g. In some embodiments, the therapeutically effective amount of benzbromarone is administered between about every 12 to about every 24 hours. In some embodiments, the therapeutically effective amount of benzbromarone is administered about every 24 hours.
[0059] In certain embodiments, the thrombotic disorder is a thrombotic disease, cardiovascular disease, procoagulant endothelial cell dysfunction, procoagulant pulmonary epithelial cell dysfunction, aberrant procoagulant activity, pathological phosphoserine extemalization, dysregulated blood coagulation, antiphospholipid antibody syndrome, SARS- CoV-2 pneumonia, thrombosis during severe SARS-CoV-2 infection, post-acute sequelae of SARS CoV-2 infection, cancer-associated thrombosis, refractory thrombosis, myocardial infarction, stroke, venous thromboembolism, disseminated intravascular coagulation (DIC), sepsis, paroxysmal nocturnal hemoglobinuria, antiphospholipid antibody syndrome, myeloproliferative disorders, inherited red cell disorders associated with thrombosis, sickle cell disease, hereditary xerocytosis, acute coronary syndrome, atrial fibrillation, or any combination thereof.
[0060] In some embodiments, the therapeutically effective amount of one or more TMEM16 inhibitors does not increase bleeding complications, bleeding time, bleeding risk, or any combination thereof. [0061] In certain embodiments of the therapeutic composition provided herein, the therapeutically effective amount of one or more TMEM16 inhibitors results in a reduction in at least one indicator of thrombotic disease, results in at least one indicator of thrombotic disease remaining within an acceptable range, or a combination thereof. In some embodiments, the at least one indicator of thrombotic disease is D-dimer, thrombin antithrombin complex, prothrombin activation peptide Fl+2, procoagulant extracellular vesicle TF cofactor activity, factor Xa generation, thrombin generation, or plasma endogenous thrombin potential. In certain embodiments, the therapeutically effective amount of one or more TMEM16 inhibitors results in concentrations of D-dimer in a patient remaining below about 500 ng/mL.
DETAILED DESCRIPTION
[0062] Before the present compositions and methods are described, it is to be understood that this invention is not limited to the particular processes, formulations, compositions, or methodologies described, as these may vary. It is also to be understood that the terminology used in the description is for the purpose of describing the particular versions or embodiments only, and is not intended to limit the scope of embodiments herein which will be limited only by the appended claims. Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of embodiments herein, the preferred methods, devices, and materials are now described. All publications mentioned herein are incorporated by reference in their entirety. Nothing herein is to be construed as an admission that embodiments herein is not entitled to antedate such disclosure by virtue of prior invention.
[0063] It must also be noted that as used herein and in the appended claims, the singular forms “a,” “an,” and “the” include plural reference unless the context clearly dictates otherwise.
[0064] As used herein, the term “about” means plus or minus 10% of the numerical value of the number with which it is being used. Therefore, about 50% means in the range of 45%-55%. [0065] The terms "treat," "treated," or "treating" as used herein refers to both therapeutic treatment and prophylactic or preventative measures, wherein the object is to inhibit, prevent or slow down or reduce the full effect or likelihood of (lessen) any undesired physiological condition, disorder or disease, or to improve, inhibit, or otherwise obtain beneficial or desired clinical results. For the purposes of this invention, beneficial or desired clinical results include, but are not limited to, improvement or alleviation of symptoms; diminishment of the extent of the condition, disorder or disease; stabilization (i.e., not worsening) of the state of the condition, disorder or disease; delay in onset or slowing of the progression of the condition, disorder or disease; amelioration of the condition, disorder or disease state; and remission (whether partial or total), whether detectable or undetectable, or enhancement or improvement of the condition, disorder or disease. Treatment includes eliciting a clinically significant response without excessive levels of side effects. Treatment also includes prolonging survival as compared to expected survival if not receiving treatment.
[0066] As used herein, the term “effective amount” refers to an amount that results in measurable inhibition of at least one symptom or parameter of a specific disorder or pathological process. As used herein, the term “therapeutically effective amount” of compositions of the application is an amount which confers a therapeutic effect on the treated subject at a reasonable benefit/risk ratio applicable to any medical treatment. The therapeutic effect may be objective (that is, measurable by some test or marker) or subjective (that is, subject gives an indication of or feels an effect, or physician observes a change).
[0067] The term “therapeutically effective amount” of an active agent, as used herein, means an amount effective, when administered to a patient, to provide a therapeutic benefit such as a prevention, inhibition, or an amelioration of symptoms, e.g., to prevent the activation of mast cells and prevent the formation of mast cell activated cytokines in a patient suffering from amyotrophic lateral sclerosis (ALS). A therapeutically effective amount may vary according to factors such as the health, age, and weight of the patient, and the ability of the compound to elicit a desired response in the patient. Dosage regimens may be adjusted to provide the optimum therapeutic response. A therapeutically effective amount is also one in which any toxic or detrimental effects (e.g., side effects) of the active agent are outweighed by the therapeutically beneficial effects.
[0068] The term “synergistic effect” as used herein, refers to an interaction or cooperation giving rise to a whole that is greater than the simple sum of its parts. As used herein, the effect of benzbromarone and isoquercetin and/or zafirlukast produces results that are greater than either benzbromarone alone. [0069] The term “patient” as used herein includes, but is not limited to, humans and nonhuman vertebrates such as wild, domestic, and farm animals. In certain embodiments, the subject described herein is an animal. In certain embodiments, the subject is a mammal. In certain embodiments, the subject is a human. In certain embodiments, the subject is a non-human animal. In certain embodiments, the subject is a non-human mammal. In certain embodiments, the subject is a domesticated animal, such as a dog, cat, cow, pig, horse, sheep, or goat. In certain embodiments, the subject is a companion animal such as a dog or cat. In certain embodiments, the subject is a livestock animal such as a cow, pig, horse, sheep, or goat. In certain embodiments, the subject is a zoo animal. In another embodiment, the subject is a research animal such as a rodent, dog, or non-human primate. In certain embodiments, the subject is a non-human transgenic animal such as a transgenic mouse or transgenic pig.
[0070] By "pharmaceutically acceptable excipients", it is meant the carrier, diluent or excipient must be compatible with the other ingredients of the topical formulation and not deleterious to the recipient thereof.
[0071] The term "thrombotic disorder" refers to many distinct conditions that cause or increase the risk of a venous or arterial thrombotic event, including but not limited to, atrial fibrillation, thrombosis due to a mechanical heart valve, myocardial infarction, unstable angina, deep vein thrombosis, acute ischemic stroke, pulmonary embolism, atherosclerosis, factor V Leiden, antithrombin III deficiency, protein C deficiency, protein S deficiency, prothrombin gene mutation (G20210A), hyperhomocysteinemia, antiphospholipid antibody syndrome, anticardiolipin antibody, thrombosis syndrome, lupus anticoagulant syndrome, malignancy, major surgery, immobilization, oral contraceptive use, thalidomide use, especially in combination with dexamethasone, heparin-induced thrombocytopenia, pregnancy, myeloproliferative disorders, inflammatory bowel disease, nephrotic syndrome, paroxysmal nocturnal hemoglobinuria, hyperviscosity syndrome, Waldenstrom's macroglobulinemia, and trauma. The term "thrombotic disorder" also refers to thrombosis induced by cancer, e.g., multiple myeloma and other hematologic cancers, adenocarcinoma, cancer of the pancreas, stomach, ovaries, prostate, colon, lung, brain, breast, kidney, skin, cervix, and ear- nose- throat cancer.
[0072] The term "blood thinning medication" refers to an antiplatelet drug, e.g., clopidogrel bisulfate, heparin, warfarin, enoxaparin, abciximab, eptifibatide, tirofiban, prasugrel, ticlopidine, beraprost, prostacyclin, iloprost, treprostinil, aspirin, aloxiprin, carbasalate calcium, indobufen, triflusal, dipyridamole, picotamide, terutroban, cilostazol, cloricromen, ditazole; or an anticoagulant, e.g., acenocoumarol, coumatetralyl, dicoumarol, ethyl biscoumacetate, phenprocoumon, clorindione, diphenadione, phenindione, tioclomarol, bemiparin, certoparin, dalteparin, nadroparin, pamaparin, reviparin, tinzaparin, fondaparinux, idraparinux, danaparoid, sulodexide, dermatan sulfate, apixaban, betrixaban, edoxaban, otamixaban, rivaroxaban, bivalirudin, lepirudin, desirudin, argatroban, dabigatran, melagatran, ximelagatran, regimen 1 (REG1; a combination of RB-006, a Factor IXa antagonist, and its oligonucleotide active control agent RB-007), defibrotide, ramatroban, antithrombin III, or drotrecogin alfa.
[0073] The term "isoquercetin" refers to certain active compounds for administration as described herein.
[0074] Isoquercetin (2-(3,4-Dihydroxyphenyl)-5,7-dihydroxy-3-[(2S,3R,4S,5S,6R)-3,4,5- trihydroxy-6-(hydroxymethyl)oxan-2-yl]oxychromen-4-one) is a 3-O-glucoside of quercetin having the following structure:
[0075] Quercetin is characterized by the following structure:
[0076] In embodiments described herein, the active compound may encompass quercetin or quercetin derivatives such as: quercetin-5-O-glucoside, quercetin-7-O-glucoside, quercetin-9- O-glucoside, quercetin-3-O-[.alpha.-rhamnosyl-(l ,fwdarw.2)-.alpha.-rhamnosyl-(l .fwdarw- .6)]- . beta. -glucoside, quercetin-3-O-galactoside, quercetin-7-O-galactoside, quercetin-3-O- rhamnoside, isoquercetin, rutin, and quercetin-7-O-galactoside. After digestion, quercetin derivatives are converted in the body to quercetin aglycon and/or other active derivatives, including methylated, sulphated and gluconorated forms which are absorbed in the body. In any embodiment described herein, the quercetin or quercetin derivative can be added to the composition either in a pure form or as an ingredient in a mixture (e.g., a plant extract). Examples of commercially available quercetin include QU995 (containing 99.5% quercetin) and QU985 (containing 98.5% quercetin) from Quercegen Pharmaceuticals LLC (Boston, Mass.).
[0077] In some embodiments described herein, one of the TMEM16 inhibitors for use in the present methods is isoquercetin. Isoquercetin can also be quercetin or derivatives of isoquercetin. In any embodiment described herein, the isoquercetin derivative can be added to the composition either in a pure form or as an ingredient in a mixture (e.g., a plant extract). Examples of commercially available isoquercetin compounds include those available from Quercegen Pharmaceuticals LLC: ISQ 995 AN (99.5% pure all-natural isoquercetin) and ISQ 995 CIT (99.5% pure isoquercitrin). Additional methods and isoquercetin compositions can be found in U.S. Pat. Nos. 7,745,486 and 7,745,487, incorporated herein by reference.
[0078] The therapeutic composition of at least two TMEM16 inhibitors may include benzbromarone, isoquercetin, zafirlukast, or any combination thereof. According to any embodiment described herein, the therapeutic composition may be administered by oral or parenteral (e.g., intramuscular, intraperitoneal, intravenous, ICV, intracistemal injection or infusion, subcutaneous injection, or implant) dosage form and may be formulated, alone or together, in suitable dosage unit formulations containing conventional non-toxic pharmaceutically acceptable carriers, adjuvants and vehicles appropriate for each route of administration. The compounds and compositions described herein may also be formulated as a controlled-release formulation.
[0079] The therapeutic composition, according to any embodiment described herein can be administered in a wide range of dosage-forms including, for example, solid dosage forms and liquid dosage forms. Solid dosage forms may include powders, tablets, pills, capsules, suppositories, or dispersible granules. A solid carrier can be one or more substances that function as a diluting agent, flavor additive, solvent, lubricant, suspension agent, binder, preservative, tablet-disintegrating substance or encapsulating material. In powdered form, the carrier may be a finely pulverized solid including lactose, hydroxypropyl methylcellulose and PVP, mixed with an appropriate amount of the active ingredient. Appropriate carriers for powder and tablet forms include for example magnesium carbonate, magnesium stearate, talc, sugar, lactose, pectin, dextrin, stiffeners, gelatins, tragacanth, methylcellulose, and sodium carboxymethylcellulose.
[0080] Liquid dosage forms include for example solutions, suspensions, and emulsions. Also included are compositions in solid form that are meant to be converted to liquid form shortly prior to consumption. These forms may include, in addition to the active ingredients, artificial colors, flavors, stabilizers, buffers, natural or artificial sweeteners, dispersing agents, thickeners, dissolving agents and the like.
[0081] Solutions or mixtures may be administered directly to the nasal cavity using conventional means, such as drops or sprays. The composition may be produced in individual or multi-dose forms. Multi-dose forms would include a dropper, pipette or atomizer that delivers a predetermined volume of the composition.
[0082] The therapeutic composition, according to any embodiment described herein may be provided in individual dosage units that contain a suitable amount of the active ingredient.
[0083] The individual doses may be provided in a package, or as a kit that includes a measuring device, e.g., a device for measuring oral or injectable dosages (i.e., a measuring cup, needle, or syringe). The kit can also include, other materials such buffers, diluents, filters, and package inserts with instructions for use. A label may be present on the on the kit to indicate that the composition is used for a specific therapy, and may also indicate directions for use.
[0084] If desired, the therapeutic compositions of the present invention may further comprise one or more additional active agents. Where it is appropriate, any of the active agents may be administered in the form of the compound per se, and/or in the form of a salt, polymorph, ester, amide, prodrug, derivative, or the like, provided the salt, polymorph, ester, amide, prodrug or derivative is suitable pharmacologically. Where it is appropriate, salts, esters, amides, prodrugs and other derivatives of the active agents may be prepared using standard procedures known to those skilled in the art of synthetic organic chemistry and described, for example, by J. March, Advanced Organic Chemistry: Reactions, Mechanisms and Structure, 4th Ed. (New York: Wiley- Interscience, 1992). For any active agents that may exist in enantiomeric forms, the active agent may be incorporated into the present compositions either as the racemate or in enantiomerically enriched form.
[0085] In some embodiments the dosage of the therapeutic composition, according to any embodiment described being administered will depend on the condition being treated, the particular compound, and other clinical factors such as age, sex, weight, and health of the subject being treated, the route of administration of the compound(s), and the type of composition being administered (tablet, gel cap, capsule, solution, suspension, inhaler, aerosol, elixir, lozenge, injection, patch, ointment, cream, etc.). It is to be understood that the present disclosure has application for both human and animal use. The amount of the therapeutic composition according to any embodiment described, required for use in treatment will be ultimately at the discretion of the attendant physician or clinician.
[0086] The therapeutic composition administered in the methods of this invention can be a sterile injectable or infusible solution that contains the therapeutic composition together with pharmaceutically acceptable excipients. Pharmaceutically acceptable excipients may include, but are not limited to, buffering agents, coloring agents, diluents, di si nt egrants, emulsifiers, flavorants, glidants, lubricants, preservatives, stabilizers, surfactants, tableting agents, and wetting agents. Some excipients may be listed in more than one class, for example vegetable oil may be used as a lubricant in some formulations and a diluent in others. Exemplary pharmaceutically acceptable excipients include sugars, starches, celluloses, powdered tragacanth, malt, gelatin, talc, and vegetable oils. Optional additional active agents may be included in a pharmaceutical composition, which do not substantially interfere with the activity of the active agent. The amount of excipient employed in conjunction with the compound is sufficient to provide a practical quantity of material for administration per unit dose of the active agent.
[0087] The dosage of the active ingredients in the therapeutic compositions of this invention may be varied so that a suitable dosage form is obtained. The active ingredients may be administered to patients (animals and human) in need of such treatment in dosages that will provide optimal pharmaceutical efficacy. The selected dosage depends upon the desired therapeutic effect, on the route of administration, and on the duration of the treatment. The dose will vary from patient to patient depending upon the nature and severity of disease, the patient's weight, special diets then being followed by a patient, concurrent medication, and other factors which those skilled in the art will recognize.
[0088] In some embodiments, the therapeutically effective amount of benzbromarone will be about 50 mg to about 800 mg daily. In certain additional embodiments, the therapeutically effective amount will be about 50 mg, 100 mg, or 200 mg daily. In certain embodiments, the therapeutically effective amount will be about 50 mg to about 500 mg daily.
[0089] In some embodiments the therapeutically effective amount of isoquercetin is between a lower limit of about 50 mg/day, about 52 mg/day, about 55 mg/day, about 57 mg/day, about 60 mg/day, about 62 mg/day, about 65 mg/day, about 67 mg/day, about 70 mg/day, about 72 mg/day, about 75 mg/day, about 77 mg/day, about 80 mg/day, about 82 mg/day, about 85 mg/day, about 87 mg/day, about 90 mg/day, about 92 mg/day, about 95 mg/day, about 97 mg/day, about 100 mg/day, about 102 mg/day, about 105 mg/day, about 107 mg/day, about 110 mg/day, 112 mg/day, about 115 mg/day, about 117 mg/day, about 120 mg/day, 122 mg/day, about 125 mg/day, about 127 mg/day, about 130 mg/day, 132 mg/day, about 135 mg/day, about 137 mg/day, about 140 mg/day, 142 mg/day, about 145 mg/day, about 147 mg/day, about 150 mg/day, about 152 mg/day, about 155 mg/day, about 157 mg/day, about 160 mg/day, about 162 mg/day, about 165 mg/day, about 167 mg/day, about 170 mg/day, about 172 mg/day, about 175 mg/day, about 177 mg/day, about 180 mg/day, about 182 mg/day, about 185 mg/day, about 187 mg/day, about 190 mg/day, about 192 mg/day, about 195 mg/day, about 197 mg/day, about 200 mg/day, about 202 mg/day, about 205 mg/day, about 207 mg/day, about 210 mg/day, 212 mg/day, about 215 mg/day, about 217 mg/day, about 220 mg/day, 222 mg/day, about 225 mg/day, about 227 mg/day, about 230 mg/day, 232 mg/day, about 235 mg/day, about 237 mg/day, about 240 mg/day, 242 mg/day, about 245 mg/day, about 247 mg/day, about 250 mg/day, about 252 mg/day, about 255 mg/day, about 257 mg/day, about 260 mg/day, about 262 mg/day, about 265 mg/day, about 267 mg/day, about 270 mg/day, about 272 mg/day, about 275 mg/day, about 277 mg/day, about 280 mg/day, about 282 mg/day, about 285 mg/day, about 287 mg/day, about 290 mg/day, about 292 mg/day, about 295 mg/day, about 297 mg/day, about 300 mg/day, about 302 mg/day, about 305 mg/day, about 307 mg/day, about 310 mg/day, 312 mg/day, about 315 mg/day, about 317 mg/day, about 320 mg/day, 322 mg/day, about 325 mg/day, about 327 mg/day, about 330 mg/day, 332 mg/day, about 335 mg/day, about 337 mg/day, about 340 mg/day, 342 mg/day, about 345 mg/day, about 347 mg/day, about 350 mg/day, about 352 mg/day, about 355 mg/day, about 357 mg/day, about 360 mg/day, about 362 mg/day, about 365 mg/day, about 367 mg/day, about 370 mg/day, about 372 mg/day, about 375 mg/day, about 377 mg/day, about 380 mg/day, about 382 mg/day, about 385 mg/day, about 387 mg/day, about 390 mg/day, about 392 mg/day, about 395 mg/day, about 397 mg/day, about 400 mg/day, about 402 mg/day, about 405 mg/day, about 407 mg/day, about 410 mg/day, 412 mg/day, about 415 mg/day, about 417 mg/day, about 420 mg/day, 422 mg/day, about 425 mg/day, about 427 mg/day, about 430 mg/day, 432 mg/day, about 435 mg/day, about 437 mg/day, about 440 mg/day, 442 mg/day, about 445 mg/day, about 447 mg/day, about 450 mg/day, about 452 mg/day, about 455 mg/day, about 457 mg/day, about 460 mg/day, about 462 mg/day, about 465 mg/day, about 467 mg/day, about 470 mg/day, about 472 mg/day, about 475 mg/day, about 477 mg/day, about 480 mg/day, about 482 mg/day, about 485 mg/day, about 487 mg/day, about 490 mg/day, about 492 mg/day, about 495 mg/day, about 497 mg/day, and about 500 mg/day; and an upper limit of about 500 mg/day, about 497 mg/day, about 495 mg/day, about 492 mg/day, about 490 mg/day, about 487 mg/day, about 485 mg/day, about 482 mg/day, about 480 mg/day, about 477 mg/day, about 475 mg/day, about 472 mg/day, about 470 mg/day, about 467 mg/day, about 465 mg/day, about 462 mg/day, about 460 mg/day, about 457 mg/day, about 455 mg/day, about 452 mg/day, about 450 mg/day, 447 mg/day, about 445 mg/day, about 442 mg/day, about 440 mg/day, about 437 mg/day, about 435 mg/day, about 432 mg/day, about 430 mg/day, about 427 mg/day, about 425 mg/day, about 422 mg/day, about 420 mg/day, about 417 mg/day, about 415 mg/day, about 412 mg/day, about 410 mg/day, about 407 mg/day, about 405 mg/day, about 402 mg/day, about 400 mg/day, 3975 mg/day, about 395 mg/day, about 392 mg/day, about 390 mg/day, about 387 mg/day, about 385 mg/day, about 382 mg/day, about 380 mg/day, about 377 mg/day, about 375 mg/day, about 372 mg/day, about 370 mg/day, about 367 mg/day, about 365 mg/day, about 362 mg/day, about 360 mg/day, about 357 mg/day, about 355 mg/day, about 352 mg/day, about 350 mg/day, 347 mg/day, about 345 mg/day, about 342 mg/day, about 340 mg/day, about 337 mg/day, about 335 mg/day, about 332 mg/day, about 330 mg/day, about 327 mg/day, about 325 mg/day, about 322 mg/day, about 320 mg/day, about 317 mg/day, about 315 mg/day, about 312 mg/day, about 310 mg/day, about 307 mg/day, about 305 mg/day, about 302 mg/day, about 300 mg/day, 297 mg/day, about 295 mg/day, about 292 mg/day, about 290 mg/day, about 287 mg/day, about 285 mg/day, about 282 mg/day, about 280 mg/day, about 277 mg/day, about 275 mg/day, about 272 mg/day, about 270 mg/day, about 267 mg/day, about 265 mg/day, about 262 mg/day, about 260 mg/day, about 257 mg/day, about 255 mg/day, about 252 mg/day, about 250 mg/day, 247 mg/day, about 245 mg/day, about 242 mg/day, about 240 mg/day, about 237 mg/day, about 235 mg/day, about 232 mg/day, about 230 mg/day, about 227 mg/day, about 225 mg/day, about 222 mg/day, about 220 mg/day, about 217 mg/day, about 215 mg/day, about 212 mg/day, about 210 mg/day, about 207 mg/day, about 205 mg/day, about 202 mg/day, about 200 mg/day, 197 mg/day, about 195 mg/day, about 192 mg/day, about 190 mg/day, about 187 mg/day, about 185 mg/day, about 182 mg/day, about 180 mg/day, about 177 mg/day, about 175 mg/day, about 172 mg/day, about 170 mg/day, about 167 mg/day, about 165 mg/day, about 162 mg/day, about 160 mg/day, about 157 mg/day, about 155 mg/day, about 152 mg/day, about 150 mg/day, 147 mg/day, about 145 mg/day, about 142 mg/day, about 140 mg/day, about 137 mg/day, about 135 mg/day, about 132 mg/day, about 130 mg/day, about 127 mg/day, about 125 mg/day, about 122 mg/day, about 120 mg/day, about 1 17 mg/day, about 115 mg/day, about 112 mg/day, about 110 mg/day, about 107 mg/day, about 105 mg/day, about 102 mg/day, about 100 mg/day, about 97 mg/day, about 95 mg/day, about 92 mg/day, about 90 mg/day, about 87 mg/day, about 85 mg/day, about 82 mg/day, about 80 mg/day, about 77 mg/day, about 75 mg/day, about 72 mg/day, about 70 mg/day, about 67 mg/day, about 65 mg/day, about 62 mg/day, about 60 mg/day, about 57 mg/day, about 55 mg/day, about 52 mg/day, and about 50 mg/day.
[0090] In some embodiments, the therapeutically effective amount of isoquercetin will be about 500 mg to up to 5 grams daily. In certain additional embodiments, the therapeutically effective amount will be about 4 grams, or 3 grams, or even 2 grams. In certain embodiments, the therapeutically effective amount will be about 500 mg to about 2000 mg daily.
[0091] In some embodiments the therapeutically effective amount of isoquercetin is between a lower limit of about 500 mg/day, about 525 mg/day, about 550 mg/day, about 575 mg/day, about 600 mg/day, about 625 mg/day, about 650 mg/day, about 675 mg/day, about 700 mg/day, about 725 mg/day, about 750 mg/day, about 775 mg/day, about 800 mg/day, about 825 mg/day, about 850 mg/day, about 875 mg/day, about 900 mg/day, about 925 mg/day, about 950 mg/day, about 975 mg/day, about 1000 mg/day, about 1025 mg/day, about 1050 mg/day, about
1075 mg/day, about 1100 mg/day, 1125 mg/day, about 1150 mg/day, about 1175 mg/day, about 1200 mg/day, 1225 mg/day, about 1250 mg/day, about 1275 mg/day, about 1300 mg/day, 1325 mg/day, about 1350 mg/day, about 1375 mg/day, about 1400 mg/day, 1425 mg/day, about 1450 mg/day, about 1475 mg/day, about 1500 mg/day, about 1525 mg/day, about 1550 mg/day, about 1575 mg/day, about 1600 mg/day, about 1625 mg/day, about 1650 mg/day, about 1675 mg/day, about 1700 mg/day, about 1725 mg/day, about 1750 mg/day, about 1775 mg/day, about 1800 mg/day, about 1825 mg/day, about 1850 mg/day, about 1875 mg/day, about 1900 mg/day, about 1925 mg/day, about 1950 mg/day, about 1975 mg/day, about 2000 mg/day, about 2025 mg/day, about 2050 mg/day, about 2075 mg/day, about 2100 mg/day, 2125 mg/day, about 2150 mg/day, about 2175 mg/day, about 2200 mg/day, 2225 mg/day, about 2250 mg/day, about 2275 mg/day, about 2300 mg/day, 2325 mg/day, about 2350 mg/day, about 2375 mg/day, about 2400 mg/day, 2425 mg/day, about 2450 mg/day, about 2475 mg/day, about 2500 mg/day, about 2525 mg/day, about 2550 mg/day, about 2575 mg/day, about 2600 mg/day, about 2625 mg/day, about 2650 mg/day, about 2675 mg/day, about 2700 mg/day, about 2725 mg/day, about 2750 mg/day, about 2775 mg/day, about 2800 mg/day, about 2825 mg/day, about 2850 mg/day, about 2875 mg/day, about 2900 mg/day, about 2925 mg/day, about 2950 mg/day, about 2975 mg/day, about 3000 mg/day, about 3025 mg/day, about 3050 mg/day, about 3075 mg/day, about 3100 mg/day, 3125 mg/day, about 3150 mg/day, about 3175 mg/day, about 3200 mg/day, 3225 mg/day, about 3250 mg/day, about 3275 mg/day, about 3300 mg/day, 3325 mg/day, about 3350 mg/day, about 3375 mg/day, about 3400 mg/day, 3425 mg/day, about 3450 mg/day, about 3475 mg/day, about 3500 mg/day, about 3525 mg/day, about 3550 mg/day, about 3575 mg/day, about 3600 mg/day, about 3625 mg/day, about 3650 mg/day, about 3675 mg/day, about 3700 mg/day, about 3725 mg/day, about 3750 mg/day, about 3775 mg/day, about 3800 mg/day, about 3825 mg/day, about 3850 mg/day, about 3875 mg/day, about 3900 mg/day, about 3925 mg/day, about 3950 mg/day, about 3975 mg/day, about 4000 mg/day, about 4025 mg/day, about 4050 mg/day, about 4075 mg/day, about 4100 mg/day, 4125 mg/day, about 4150 mg/day, about 4175 mg/day, about 4200 mg/day, 4225 mg/day, about 4250 mg/day, about 4275 mg/day, about 4300 mg/day, 4325 mg/day, about 4350 mg/day, about 4375 mg/day, about 4400 mg/day, 4425 mg/day, about 4450 mg/day, about 4475 mg/day, about 4500 mg/day, about 4525 mg/day, about 4550 mg/day, about 4575 mg/day, about 4600 mg/day, about 4625 mg/day, about 4650 mg/day, about 4675 mg/day, about 4700 mg/day, about 4725 mg/day, about 4750 mg/day, about 4775 mg/day, about 4800 mg/day, about 4825 mg/day, about 4850 mg/day, about 4875 mg/day, about 4900 mg/day, about 4925 mg/day, about 4950 mg/day, about 4975 mg/day, and about 5000 mg/day; and an upper limit of about 5000 mg/day, about 4975 mg/day, about 4950 mg/day, about 4925 mg/day, about 4900 mg/day, about 4875 mg/day, about 4850 mg/day, about 4825 mg/day, about 4800 mg/day, about 4775 mg/day, about 4750 mg/day, about 4725 mg/day, about 4700 mg/day, about 4675 mg/day, about 4650 mg/day, about 4625 mg/day, about 4600 mg/day, about 4575 mg/day, about 4550 mg/day, about 4525 mg/day, about 4500 mg/day, 4475 mg/day, about 4450 mg/day, about 4425 mg/day, about 4400 mg/day, about 4375 mg/day, about 4350 mg/day, about 4325 mg/day, about 4300 mg/day, about 4275 mg/day, about 4250 mg/day, about 4225 mg/day, about 4200 mg/day, about 4175 mg/day, about 4150 mg/day, about 4125 mg/day, about 4100 mg/day, about 4075 mg/day, about 4050 mg/day, about 4025 mg/day, about 4000 mg/day, 3975 mg/day, about 3950 mg/day, about 3925 mg/day, about 3900 mg/day, about 3875 mg/day, about 3850 mg/day, about 3825 mg/day, about 3800 mg/day, about 3775 mg/day, about 3750 mg/day, about 3725 mg/day, about 3700 mg/day, about 3675 mg/day, about 3650 mg/day, about 3625 mg/day, about 3600 mg/day, about 3575 mg/day, about 3550 mg/day, about 3525 mg/day, about 3500 mg/day, 3475 mg/day, about 3450 mg/day, about 3425 mg/day, about 3400 mg/day, about 3375 mg/day, about 3350 mg/day, about 3325 mg/day, about 3300 mg/day, about 3275 mg/day, about 3250 mg/day, about 3225 mg/day, about 3200 mg/day, about 3175 mg/day, about 3150 mg/day, about 3125 mg/day, about 3100 mg/day, about 3075 mg/day, about 3050 mg/day, about 3025 mg/day, about 3000 mg/day, 2975 mg/day, about 2950 mg/day, about 2925 mg/day, about 2900 mg/day, about 2875 mg/day, about 2850 mg/day, about 2825 mg/day, about 2800 mg/day, about 2775 mg/day, about 2750 mg/day, about 2725 mg/day, about 2700 mg/day, about 2675 mg/day, about 2650 mg/day, about 2625 mg/day, about 2600 mg/day, about 2575 mg/day, about 2550 mg/day, about 2525 mg/day, about 2500 mg/day, 2475 mg/day, about 2450 mg/day, about 2425 mg/day, about 2400 mg/day, about 2375 mg/day, about 2350 mg/day, about 2325 mg/day, about 2300 mg/day, about 2275 mg/day, about 2250 mg/day, about 2225 mg/day, about 2200 mg/day, about 2175 mg/day, about 2150 mg/day, about 2125 mg/day, about 2100 mg/day, about 2075 mg/day, about 2050 mg/day, about 2025 mg/day, about 2000 mg/day, 1975 mg/day, about 1950 mg/day, about 1925 mg/day, about 1900 mg/day, about 1875 mg/day, about 1850 mg/day, about 1825 mg/day, about 1800 mg/day, about 1775 mg/day, about 1750 mg/day, about 1725 mg/day, about 1700 mg/day, about 1675 mg/day, about 1650 mg/day, about 1625 mg/day, about 1600 mg/day, about 1575 mg/day, about 1550 mg/day, about 1525 mg/day, about 1500 mg/day, 1475 mg/day, about 1450 mg/day, about 1425 mg/day, about 1400 mg/day, about 1375 mg/day, about 1350 mg/day, about 1325 mg/day, about 1300 mg/day, about 1275 mg/day, about 1250 mg/day, about 1225 mg/day, about 1200 mg/day, about 1175 mg/day, about 1150 mg/day, about 1125 mg/day, about 1100 mg/day, about 1075 mg/day, about 1050 mg/day, about 1025 mg/day, about 1000 mg/day, about 975 mg/day, about 950 mg/day, about 925 mg/day, about 900 mg/day, about 875 mg/day, about 850 mg/day, about 825 mg/day, about 800 mg/day, about 775 mg/day, about 750 mg/day, about 725 mg/day, about 700 mg/day, about 675 mg/day, about 650 mg/day, about 625 mg/day, about 600 mg/day, about 575 mg/day, about 550 mg/day, about 525 mg/day, and about 500 mg/day
[0092] The compounds may be administered on a regimen of 1 to 4 times per day, such as once, twice, three times or four times per day.
[0093] The coronavirus that causes COVID-19, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), can lead to hypercoagulation in a patient due to inflammation in endothelial and epithelial tissue. The tissue affected can be in the lung, heart, veins, arteries, or other tissues. Thrombosis related to COVID-19 can be detected as pulmonary embolism, large- vessel thrombosis, stroke, thromboses in blood vessels that supply the small intestine, obstructions in peripheral and large vessel arteries, and cerebral venous sinus thrombosis. Microvascular inflammation and subsequent microthrombosis can lead to tissue damage, with long-term negative effects. COVID-19 related hypercoagulation may be related to many physiological responses, including complement activation, the renin-angiotensin system, platelet activation, or a cytokine storm. Treatment of hypercoagulation in COVID-19 commonly uses doses of heparin; the present disclosure provides a method of treating COVID- 19 related thrombosis or hypercoagulation with TMEM16 inhibitors, such as benzbromarone. In some embodiments, when benzbromarone is administered in combination with isoquercetin, it can produce a synergistic effect, including but not limited to, increased protection against micro and macro thrombosis, decreased platelet aggregation, decreased fibrin formation, and lowered levels of P-dimer and other indicators of thrombosis.
[0094] Post-acute sequelae of SARS CoV-2 infection, or long COVID, occurs when a patient does not recover within the usual one or two weeks after contracting COVID-19. Long COVID leads to a wide variety of symptoms, with multiorgan effects, and is difficult to diagnose and treat. There is an increased propensity for hypercoagulation in long COVID, and more treatments are needed to prevent the increased risks of damage to lungs, kidneys, and blood vessels. Treatment with TMEM16 inhibitors is useful in both preventing and lowering the risk of thrombosis in patients with long COVID, and can be accomplished through administering benzbromarone or through administering a combination of benzbromarone with one or more of isoquercetin and zafirlukast.
[0095] The efficacy of administering isoquercetin to reduce the hypercoagulability in cancer patients was evaluated (See, Zwicker et al., JCI Insight.2019; 4(4):el25851, and also Clinicaltrials.gov NCT02195232). Venous thromboembolism (VTE) is commonly observed in cancer patients and is a leading cause of mortality in this population. In high risk cancer patients especially where protocol-driven radiographic monitoring for deep vein thrombosis is implemented, the incidence of VTE within initial few months of chemotherapy commonly exceeds 20%. Cancer patients are also at an increased risk of bleeding which has limited the adoption of routine primary thromboprophylaxis in cancer outpatients receiving chemotherapy. Developing treatment methods that reduce the incidence of VTE without increasing the risk of major hemorrhage would broadly impact the care of patients with advanced malignancy, as well as any patient with cancer.
[0096] As used herein, the types of cancer can be selected from the group consisting of estrogen receptor-dependent breast cancer, estrogen receptor-independent breast cancer, hormone receptor-dependent prostate cancer, hormone receptor-independent prostate cancer, brain cancer, renal cancer, glioblastoma, colon cancer, familial adenomatous polyposis (FAP), colorectal cancer, pancreatic cancer, bladder cancer, esophageal cancer, stomach cancer, genitourinary cancer, gastrointestinal cancer, uterine cancer, ovarian cancer, astrocytomas, gliomas, skin cancer, squamous cell carcinoma, Keratoakantoma, Bowen disease, cutaneous T-Cell Lymphoma, melanoma, basal cell carcinoma, actinic keratosis; ichtiosis; acne, acne vulgaris, sarcomas, Kaposi's sarcoma, osteosarcoma, head and neck cancer, small cell lung carcinoma, non-small cell lung carcinoma, leukemia, lymphomas and/or other blood cell cancers.
[0097] Additional cancers that will benefit from the methods described herein include cancers associated with certain viruses (and include improving a pre-cancerous condition during viral infection). Such conditions include those associated with Human T-cell leukemia virus type, also called human T-lymphotrophic virus (HTLV-1) which is linked to adult T-cell leukemia/lymphoma. Another such cancer include those associated with human papillomavirus (HPV), which has at least 12 strains that can cause cancer in men and women, including anal, cervical, penile, throat, vaginal and vulvar cancer. Additional condition includes those associated with human herpes virus 8 (HHV-8), which is associated with Kaposi sarcoma in people who have a weakened immune system (e.g. patients with HIV). Similarly, there are numerous cancers associated with HIV, which is believed to damage the immune system and reduce defenses against other oncoviruses. HIV-associated cancers include Kaposi sarcoma, non-Hodgkin’s and Hodgkin’s lymphoma, cervical cancer, and cancers of the anus, liver, mouth and throat and lung. Additionally, hepatitis C is a leading cause of liver cancer, and can cause non-Hodgkin’s lymphoma, and as such can benefit from the methods described herein. Similarly, hepatitis B is a leading cause of liver cancer, and these conditions can benefit from the methods described herein. Finally, Epstein-Barr virus (EBV) infection increases the risk of Burkitt lymphoma, some types of Hodgkin’s and non-Hodgkin’s lymphoma and stomach cancer, and these conditions can also benefit from the methods described herein. [0098] In certain embodiments, the cancer is a metastasizing cancer. A "metastasizing cancer" is a cancer which may form or often forms metastases. A metastasizing cancer which has already spread from the part of the body where it started, i.e. the primary site, to other parts of the body, is also denoted metastatic cancer. When cancer cells break away from a tumor, they can travel to other areas of the body through the bloodstream or the lymph system. Such cancer cells may then form new tumors in other areas of the body.
[0099] In certain embodiments, the cancer is a metastasizing cancer selected from the group consisting of metastasizing forms of Hodgkin lymphoma, colorectal cancer, cervical cancer, lung cancer, skin cancer such as squamous cell cancer or basal cell carcinoma, head and neck cancer, gastric cancer, pancreatic cancer, head and neck squamous cell cancer, and breast cancer. [00100] In some embodiments the metastatic cancer is colorectal cancer, pancreatic cancer, or non-small cell lung cancer.
[00101] In certain embodiments, the cancer is classifiable as Stage III or Stage IV according to the TNM anatomic/prognostic group system of the cancer staging system of the American Joint Committee on Cancer. In additional embodiments, the cancer is classifiable as Stage IV according to the TNM anatomic/prognostic group system of the cancer staging system of the American Joint Committee on Cancer.
[00102] In certain embodiments, the cancer is a metastasizing cancer selected from the group consisting of metastasizing forms of Hodgkin lymphoma, colorectal cancer, cervical cancer, lung cancer, skin cancer such as squamous cell cancer or basal cell carcinoma, head and neck cancer, gastric cancer, pancreatic cancer, and breast cancer, wherein said metastasizing cancer is classifiable as Stage IV according to the TNM anatomic/prognostic group system of the cancer staging system of the American Joint Committee on Cancer (7.sup.th edition, 2010, Springer).
EXAMPLES
Example 1 - Regulation of endothelial cell procoagulant activity with TMEM16 inhibitors
Methods
Cell culture and siRNA transfection
[00103] Human umbilical vein endothelial cells (HUVEC, pooled donor, Lonza) were grown and maintained in endothelial cell growth basal media (EBM-2, Lonza), containing 2% fetal bovine serum (FBS) and contents of the EGM-2 SingleQuots growth factor supplement kit. Cells from passages 3-5 were used for experiments. EA.hy296 cells stably expressing tissue factor (EA.hy.296-TF high cells) (28) were cultured in DMEM containing 10% FBS. Cells were reverse transfected with siRNA at a final concentration of 20 pM (96-well plate) or 40 pM (384-well plate) using Lipofectamine RNAiMax (ThermoFisher Scientific) according to manufacturer protocol. The following siRNA sequences were used (Dharmacon siGENOME or ON-TARGET) TMEM16E (ANO5y. CUACGUAGCUUUCUUUAAA and GCACACUCCUAUAAGCUAU, TMEM16F (ANO6) GAUCAUCGCUUCAGUUAUU, CAACUCAGCUGACAAUAAU, TF (F3) CAUUGGAGCUGUGGUAUUU; Stiml (STIM1) siGENOME SMARTpool siRNA: CAUCAGAAGUAUACAAUUG, AGAAGGAGCUAGAAUCUCA,
AGGUGGAGGUGCAAUAUUA, and GGUGGUGUCUAUCGUUAUU.
Factor Xa and thrombin generation assays
[00104] Primary HUVECs or EA.hy296-TF high cells were plated in 384-well or gelatin- coated 96-well plates. For experiments using siRNA transfection, 1,000 (384-well plate) or 5,000 (96-well plate) cells were transfected, plated, and allowed to grow for 72 hours. For all other experiments, 20,000 cells were plated (96-well plate) one day before the experiment. Where indicated, cells were stimulated with tumor necrosis factor-a, (TNF-a, 10 ng/mL, R&D Systems) for 3.5 hours and/or Ca2+ ionophore A23187 (Sigma) for 20 min at 37°C, washed twice with HBS- BSA (20 mM HEPES, pH 7.4, 150 mM NaCl, 5 mM KC1, 5 mM CaCh, 1 mg/mL fatty acid-free bovine serum albumin, Sigma) and equilibrated to room temperature. For Factor Xa generation experiments, cells were incubated with HBS-BSA containing factor X (125 nM, Haematologic Technologies, factor Vila (0.6 nM, Haematologic Technologies), and factor Xa chromogenic substrate Biophen-CSl 1(22) (150 pM, AniaraDiagnostica). Absorbance at 405 nm was measured every minute for 3 hours on an xMark Spectrophotometer (Bio-Rad). Maximal reaction velocity was converted to factor Xa nM/min based on standard curve analysis of isolated factor Xa (Haematologic Technologies) serial dilutions with the same reaction conditions. In the focused siRNA screen of PLSs (FIG. 1A), cells were transfected with the corresponding Dharmacon siGENOME gene-specific siRNA pool in 384-well format and assayed as in the above kinetic experiments, except that factor Xa generated was determined at 45 minutes on an EnVision plate reader (Perkin Elmer) and presented as a percentage of untargeted siRNA control. For experiments using CaCCinh-AOl or benzbromarone (Cayman Chemical), drug (0.03-30 pM) or DMSO vehicle control was added at the time of TNF-a stimulation. When TF inhibitory antibody (10 pg/mL, 4509, BioMedica Diagnostics) or bovine lactadherin (100 nM, Haematologic Technologies) were evaluated, cells were washed in HBS-BSA and incubated in the presence of indicated protein in HBS-BSA for 10 min. The reaction was then triggered by addition of factors X and Vila and factor Xa subtrate at concentrations indicated above. For thrombin generation experiments, cells were washed twice with HBS-BSA and incubated in 80 pL HBS plus 20 pL pooled human plasma (George King Bio-Medical) to supply coagulation factors and 5 mM H-Gly-Pro-Arg-Pro-OH fibrin polymerization inhibitor (GPRP, 5 mM, Cayman Chemical) and fluorogenic thrombin substrate B0C-L-FPR-ANSNH-C2H5 (50 pM, SN-20, Haematologic Technologies). In certain samples, bovine lactadherin (100 pM, Haematologic Technologies) was added to the reaction mixutre. The reaction was initiated by addition of 0.8-1.0 mM CaCh and read immediately using a Synergy HTX plate reader (BioTek). Fluorescence (excitation 352 nm / emission 470 nm) was measured every minute for 1 hour. First derivative of thrombin generation curves were compared with a standard curve of thrombin to determine thrombin generated in U/mL. All individual experiments were performed in technical triplicate and the mean of these technical replicates was used as the value for each independent experiment.
Immunofluorescence microscopy
[00105] Following siRNA transfection, HUVECs were plated directly in gelatin-coated glass chamber slides for 72 hours. Cells were washed with 10 mM HEPES buffer (pH 7.4) containing 140 mM NaCl, 2.5 mM CaCh, (annexin V binding buffer) and stained with annexin V Alexa Fluor 488 (Thermo Fischer Scientific) at a 1:50 dilution and Zombie Red viability dye (BioLegend) at a 1 : 1000 dilution in the dark for 15 minutes at room temperature in annexin V binding buffer. Cells were washed and fixed in annexin V binding buffer containing 4% paraformaldehyde for 7 minutes. Cells were washed 3 times and mounted with DAPI (ProLong Gold Antifade Mountant, ThermoFisher Scientific). For tissue factor immunofluorescence staining, cells were washed 3 times with PBS and fixed in PBS containing 4% paraformaldehyde for 7 minutes. To avoid permeabilizing cells, no detergents were added in the blocking or washing steps. Cells were blocked in PBS containing 10% goat serum (Jackson ImmunoResearch) and 1% BSA for 1 hour and then stained in blocking solution containing anti-TF antibody (5 pg/mL, 4509, BioMedica Diagnostics) overnight at 4°C. Cells were washed 3 times in PBS and stained with anti-mouse secondary antibody conjugated to Alex-fluor 488 (Jackson ImmunoResearch) at a dilution of 1 :500 for 1 hour at room temperature. Cells were washed 3 times in PBS and mounted in DAPI. Images were obtained using a Zeiss LSM 880 upright laser scanning confocal microscope in 3 x 3 tile-scan mode with a Plan- Apochromat 20X/0.8 M27 objective. Fluorescent images were analyzed using Image J software (NIH). For phosphatidyl serine externalization, the threshold for annexin V staining was equalized for all images and total fluorescent area was obtained, minus Zombie Red fluorescent area and normalized to the number of nuclei. For TF immunofluorescence, mean fluorescence intensity was quantified per tissue area, after measuring and subtracting background signal from each image.
Flow cytometry
[00106] HUVECs were stimulated with indicated concentration of Ca2+ ionophore A23187 (Sigma) for 15 minutes at 37°C and washed with PBS. For experiments using CaCCinh-AOl or benzbromarone (Cayman Chemical), drug (10 pM) or DMSO vehicle control was added 90 minutes prior to A23187 stimulation. Cells were dissociated using Accutase (StemCell Technologies), and washed by re-pelleting twice in cold PBS. Cells (~1 x 106 per mb) were resuspended in 10 mM HEPES buffer (pH 7.4) containing 140 mM NaCl, 2.5 mM CaCh, and 1 mg/mL BSA and PS externalization was determined by staining with annexin V conjugated to Alexa Fluor 488 (Thermo Fischer Scientific) at a 1 :20 dilution with DAPI solution (0.1 pg/mL, BD Biosciences) as a viability stain for 15 minutes in the dark. Cells were gated using forward and side scatter and 10,000 events were collected using a CytoFLEX LX (Beckman Coulter) using CytExpert software. Data were analyzed using FloJo software (BD Biosciences, Ashland, OR), and annexin V-positive (PS externalized) cells were determined in populations of viable (D APInegative) cells only.
Intracellular calcium measurements
[00107] Confluent HUVECs were washed with HBSS (140 mM NaCl, 5 mM KC1, 1 mM CaCL, 4 mM MgSO4, 5 mM MgCE, 3 mM Na2HPO4, 4 mM KH2PO4, 6 mM D-glucose, 4 mM NaHCCf) and loaded with 100 pL of 4 pM Calbryte 520 AM (AAT Bioquest) in HBSS for 30 minutes. Calbryte 520 does not bind Ca2+ until it is esterified intracellularly and therefore can be loaded in buffers containing divalent cations. After incubation, samples were washed with HBSS and replaced with 100 pL of HBSS as reaction volume. For experiments involving drug treatment of HUVECs, the compounds were added during Calbryte 520 loading for 30 minutes. U73122 was obtained from Tocris. All Ca2+ flux assays were performed using the Molecular Devices FlexStation III dual-monochromator plate reader with automated pipetting at the Harvard ICCB- Longwood Screening Facility. Fluorescence was measured every 1.2 seconds on the FlexStation at Ex/Em = 490/525 nm and a cutoff of 515 nm. After an initial baseline fluorescence reading of 30 seconds, cells were treated with human alpha-thrombin (Haematologic Technologies) in stimulant volume of 25 pL for a final concentration of 1 U/mL. The 30 second baseline fluorescent readings were averaged (Fo) and the relative change in fluorescence was calculated according to the equation (F-Fo)/Fo. The fluorescent readings from technical triplicates of the same condition were averaged and the area under the curve was calculated using GraphPad Prism (version 9.0; GraphPad Software, San Diego, CA).
Blood and coagulation assays
[00108] Hematologic analysis of whole blood counts was performed using a Hemavet 850FS (Drew Scientific) for white blood cell counts, hemoglobin measurement, and platelet counts. The prothrombin time was measured using the Neoplastine Cl Plus (Diagnostica Stago, Inc.) and the activated partial thromboplastin time was measured using STA-PTT (Diagnostica Stago, Inc.). Coagulation assays were read on a Start benchtop analyzer (Diagnostica Stago, Inc.) according to manufacturer’s instructions.
Quantitative PCR
[00109] Gene expression was determined using a 2-step Cell-to-CT kit (Thermo Fisher Scientific) with the Hsl381106_ml (AN05), Hs03805835_ml (AN06), Hs00372436_sl and Hs00194899_ml (ACTB) gene expression probes (Taqman, Thermo Fisher Scientific). Quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) was performed in technical duplicate for each biologic sample using a QuantStudio 6 Flex real-time PCR system. Gene expression was compared to ACTB expression using the DDCt method.
Lentiviral gene transfer
[00110] Full length TMEM16E (AN05) coding sequence (Harvard Plasmid Core, clone Hs CD00345649) was subcloned into the lentiviral vector pLX304 (Addgene plasmid #25890, gift from David Root). Lenti-X 293T cells (Takara) were cotransfected with ANO5-pLX304 lentiviral transfer vector and packaging vectors (pMD2.G and psPAX2, Addgene plasmids #12259 and #12260, gift from Didier Trono) using Lipofectamine 3000 (ThermoFisher Scientific). Beginning after 48 hours, the viral supernatant was collected twice every 24 hours and replaced with fresh DMEM supplemented with 10% fetal bovine serum. Primary HUVECs (passage 1-2) were cultured in the presence of viral supernatant for 6 hours and then replaced with complete endothelial cell growth media (see Endothelial cell culture and siRNA transfection) for 48 hours before selecting for transduced cells with addition of blasticidin (10 pg/mL) to the cell culture media.
Immunoblotting
[00111] HUVEC cultures were lysed using RIPA buffer (Boston Bioproducts) supplemented with cOmplete Protease Inhibitor Cocktail (Roche) and PhosStop phosphatase inhibitor cocktail (Roche), 1 mM Na3VO4 (New England Biolabs), and 1 mM NaF. Proteins were resolved via SDS-PAGE 4-12% gradient gels (Thermofisher Scientific) under reducing conditions using NuPAGE SDS sample buffer and sample reducing agent (ThermoFisher Scientific), transferred to a nitrocellulose membrane, and blocked with SuperBlock buffer (ThermoFisher Scientific). Protein detection was performed with the following primary antibodies: TMEM16E/Ano5 (clone N421A/85, UC Davis/NIHNeuroMab), V5-Tag (#80076, Cell Signaling Technology), TMEM16F (Sigma), b-actin (#12620, Cell Signaling Technologies) and appropriate species-specific HRP-conjugated secondary antibodies (Cell Signaling Technologies). Immunoblots were developed with Supersignal West Dura or Femto Chemiluminescent Substrate (ThermoFisher Scientific) and visualized with Syngene BioImage and GeneSnap image acquisition software.
Cell viability assay
[00112] To assess cell viability of cells transfected with indicated siRNAs, an XTT Cell Proliferation Assay (ATCC) was performed according to manufacturer protocol. Absorbance at 630 nm and 450 nm were measured on a on an xMark Spectrophotometer (Bio-Rad).
Confocal intravital microscopy for Z-stack images
[00113] Wild-type C57BL/6J male mice were anesthetized and prepped as described in “Intravital microscopy and laser-induced vessel wall injury model” and the cremaster arteriole was injured with a laser ablation with (Ablate!, 3i). Z-stack images were obtained on an CSU-W1 spinning disk confocal microscope with SoRa super resolution optical unit (Yokagawa) using a 6-line laser illumination system (3i), 63X high numerical aperture water corrected lens (Zeiss) and Ocra Fusion BT sCMOS digital video camera (Hamamatsu). Z-stack images were rendered into 3-dimensional images using using Slidebook version 6.0 (Intelligent Imaging Innovations).
Intravital microscopy and laser-induced vessel wall injury model [00114] Thrombus formation was visualized via intravital microscopy following laser- induced injury to the cremasteric arteriole in male mice as previously described. Platelets were detected using anti-CD42b antibody (0.1 mg/g body weight; Emfret Analytics) conjugated to Dylight 405 (Thermo Fisher Scientific), fibrin was detected with anti-fibrin antibody (0.5 mg/g body weight; clone 59D8) conjugated to Dylight 488 (Thermo Fisher Scientific), and PS extemalization was detected using annexin V (0.025 pg/g body weight) conjugated to Alexa Fluor 647 (Thermo Fisher Scientific) infused via internal jugular vein catheter. Annexin V was re-dosed every 10-15 minutes due to its rapid metabolism. For indicated experiments, lactadherin conjugated to FITC (Haematologic Technologies) or pSIVA (Novus Biologicals) were injected intravenously at a dose of 1 pL/g body weight. The cremasteric microcirculation was surgically exposed and injury to the cremasteric arteriole was stimulated with a MicroPoint Laser System (Photonics Instruments). Imaging was performed on a M205 FCA microscope (Leica) with LEDbased fluorescence light engine (SpectraX, Lumencor). Data were digitally captured via an Orca Flash 4.0v2 CMOS camera in both the 488/520 nm and 647/670 nm fluorescence channels at a rate of 2 frames per second beginning before and extending for 180 seconds after laser injury. Images were analyzed using Slidebook version 6.0 (Intelligent Imaging Innovations). Injury size was determined using calipers one frame after laser injury (59). For each thrombus generated, a rectangular mask was defined that included a portion of the vessel upstream of the site of injury. The maximum fluorescence intensity of the pixels contained in this mask was extracted for all frames (pre- and post-injury) for each thrombus. The mean value calculated from the maximal intensity values in the mask for each frame was determined and used as the background value. Finally, for each frame the integrated fluorescence intensity was calculated as per following equation: Integrated fluorescence intensity = Sum Intensity of signal - (mean of the maximal background intensity x area of the signal). This calculation was performed for all frames in each thrombus and plotted versus time to provide the kinetics of thrombus formation. Area under the curve was calculated individual thrombi to evaluate for statistical significance (Higgins et al., J Clin Invest 128:1471-1484 (2018), Schulman et al., J Clin Invest 130:5302-5312 (2020). For experiments with eptifibatide, mice were injected intravenously with eptifibatide (10 pg/g body weight, Cayman Chemical) every 10-15 minutes. For multiple fluorescence channels, calculations of background were made independently for each channel. At least n = 30 injuries across at least 3 mice were used to determine the median value of the integrated fluorescence intensity to account for the variability of thrombus formation at any given set of experimental conditions. The operator was blinded to genotype during injury and image analysis and to drug-dosing during image analysis in all experiments.
Tail amputation bleeding assay
[00115] The tail was immersed in saline prewarmed to 37°C for 2 minutes. The tail was then amputated 5 mm from the tip and immediately immersed back into 15 mL of warmed saline. The total bleeding time (including re-bleeding time) was recorded for 10 minutes. The amount of blood loss was quantitated by measuring hemoglobin content of the blood collected in the 15 mL test tube. The red blood cells were pelleted by centrifugation (300 g for 6 minutes). The pellet was lysed in 6 mL red blood cell lysis buffer and the amount of hemoglobin was spectrophotometrically determined by measuring light absorbance at 575 nm.
Animals
[00116] TMEM16E (^nodj-null mice on a C57BL/6J background were described previously (33). Wild-type C57BL/6J mice were obtained from Jackson Laboratory (Bar Harbor, ME). Experiments were performed on mice of 8-12 weeks of age. For experiments with benzbromarone (Cayman Chemical), the compound was dissolved to a concentration of 40 pg/mL in DMSO and dissolved to a working solution in com oil to a final concentration of 1 pg/mL. Benzbromarone or DSMO vehicle control in corn oil was injected intraperitoneally at a concentration of 5 ug/g body weight 1 hour prior to experiments. Mice were anesthetized with intraperitoneal injection of ketamine (125 pg/g) and xylazine (12.5 pg/g) and secured on a heating pad via taping of the paw tips. For intravital microscopy experiments, additional IV anesthesia with pentobarbital (5 pg/g) was administered via the internal jugular catheter.
Statistics
[00117] Tests of normality were performed using the Anderson-Darling and D’Agonstino- Pearson method. Statistical significance for binary comparisons of continuous variables were assessed by unpaired two-tailed Student’s t test unless the data did not demonstrate normality, which was the case for intravital imaging data, in which case differences between groups were analyzed by Mann-Whitney U test as previously described (Higgins et al., J Clin Invest 128:1471- 1484 (2018); Ivanciu et al., Blood 124: 1705-1714 (2014); Schulman et al., J Clin Invest 130:5302- 5312 (2020)). For comparison of more than 2 groups, one-way analysis of variance was performed with application of Tukey’s posttest method to adjust for multiple comparisons. Assumptions on the statistical power for rodent experiments was based on our extensive experience with the performance characteristics of the intravital cremasteric laser injury model and the tail clip bleeding assay. All statistical analysis was performed using GraphPad Prism (version 9.0; GraphPad Software, San Diego, CA). P values of less than 0.05 were considered significant.
Study approval
[00118] The Beth Israel Deaconess Medical Center Institutional Animal Care and Use Committee approved all animal care and experimental procedures.
Results
Identification of transmembrane lipid transporters predicted to regulate EC procoagulant activity
[00119] Small interfering RNA (siRNA) was used to evaluate genes with established roles in PS extemalization and measured coagulation initiation on TNF-a-stimulated ECs. Primary human umbilical vein endothelial cells (HUVECs) were transfected with gene-specific pools of four distinct siRNAs and cultured for 72 hours prior to cytokine stimulation. The TNF-a-treated cells, providing the exclusive source of TF and membrane surface, were then assayed for their ability to support factor Vila catalyzed conversion of factor X to factor Xa (FIG. 1A-D). Since membrane PS composition is a critical determinant of factor X activation by the TF-VIIa complex (Bach and Rifkin, Proc Natl Acad Sci USA 87:6995-6999 (1990), Nemerson, J Clin Invest 47:72- 80 (1968), Ruf et al., J Biol Chem 1991;266:2158-2166 (1991), Wolberg et al., Blood Coagul Fibrinolysis 10:201-210 (1999)), our approach had the ability to identify regulators of anionic phospholipid membrane asymmetry that play a role in coagulation. Here 13 validated genes are studied, known to affect the outer leaflet expression of PS in biological membranes, including six members of the TMEM16 (gene name, ANO) family of Ca2+-activated PLSs, three members of the Xk-related (XKR) family of apoptotic PLSs (Suzuki et al., J Biol Chem 289:30257-30267 (2014)), and three members of the P4-ATPase family of phospholipid flippases (Segawa et al., J Biol Chem 291 :762-772 (2016)), including its cofactor CDC50A (gene name TMEM30A). TMEM16E (AN05) was identified as the most significant positive regulator of factor Vila- catalyzed procoagulant activity in ECs (FIG. 1A). TMEM16E is the closest paralog of the canonical Ca2+-activated PLS,TMEM16F (AN06) (Whitlock and Hartzell, Annu Rev Physiol 79: 119-143 (2017)), and TMEM16E has been shown to have PLS activity (Whitlock et al., J Gen Physiol 2018;150: 1498-1509 (2018), Gyobu et al., Mol Cell Biol 36:645-659 (2016)). TMEM16F was also identified as a positive regulator of EC procoagulant activity (FIG. 1A). TMEM16E and TMEM16F as PLSs that may promote coagulation on ECs.
TMEM16E and TMEM16F are required for procoagulant activity in ECs
[00120] To validate these findings, the requirement of TMEM16E and TMEM16F in supporting coagulation on ECs was tested. Primary human ECs from various tissues express both TMEM16E and TMEM16F in a manner independent of TNF-a stimulation (FIG. 7A-C). Two distinct siRNAs were confirmed to antagonize expression of TMEM16E or TMEM16F in ECs (FIG. 8A-E). TMEM16E or TMEM16F were silenced in primary HUVECs and stimulated these cells with TNF-a to induce expression of TF. Cells were then tested for their ability to support factor Vila catalyzed factor Xa generation assay in a one-stage factor Xa generation assay. Silencing of either TMEM16E or TMEM16F resulted in approximately 50% reduction of TF- dependent factor Xa generation compared to non-targeting control siRNA (FIG. IB and FIG. 1C). This reduction is comparable to that observed in cells treated with the PS-binding protein lactadherin (FIG. IB and FIG. 1C). Silencing of TMEM16E or TMEM16F also inhibited TF- dependent factor Xa generation that was augmented by addition of ionophore A23187 (FIG. ID), which promotes PS externalization by raising intracellular Ca2+ (Balasubramanian et al., J Biol Chem 282: 18357-18364 (2007); Bevers et al., Biochim Biophys Acta 736:57-66 (1983)). The degree of inhibition was again similar to that observed with lactadherin, suggesting that TMEM16E and TMEM16F may promote TF-dependent procoagulant activity through PS externalization (FIG. 1C and FIG. ID). Dual silencing of TMEM16E and TMEM16F did not further suppress factor Xa generation beyond silencing of either gene product alone (FIG. 2C and FIG. 2D, see discussion). To uncouple the effects of TNF-a on TF expression and PS externalization, an Ea.hy926 cell line stably expressing TF (Higgins et al., J Clin Invest 128:1471- 1484 (2018)) was used. In Ea.hy926-TF cells, TMEM16E and TMEM16F were required for Ca2+ ionophore-induced augmentation of TF-dependent factor X activation (FIG. 9).
[00121] ECs also required TMEM16E and TMEM16F to support plasma-derived thrombin generation following TNF-a stimulation (FIG. IE and FIG. IF). Treatment with Ca2+ ionophore A23187 alone promoted thrombin generation that was inhibited following silencing of TMEM16E, TMEM16F or addition of lactadherin (FIG. 1G). In this assay, EC-mediated thrombin generation was completely abolished by lactadherin (FIG. IF and FIG. 1G). Together, these results demonstrated that both TMEM16E and TMEM16F were necessary to support procoagulant activity on the endothelial cell surface.
TMEM16E and TMEM16F regulate PS externalization on ECs
[00122] TMEM16E and TMEM16F function as Ca2+-activated PLSs, disrupting membrane phospholipid asymmetry by allowing PS and other anionic phospholipids to move down their concentration gradient from the inner to the outer membrane leaflet (Bevers and Williamson, Physiol Rev 96:605-645 (2016); Whitlock et al., J Gen Physiol 2018;150: 1498-1509 (2018); Yu et al., Elife 4: 1-23 (2015)). Indeed, PS externalization in response to TNF-a or Ca2++ ionophore was markedly inhibited in HUVECs following silencing of TMEM16E or TMEM16F as detected by annexin V binding and immunofluorescence microscopy (FIG. 2A and FIG. 2B) or flow cytometry (FIG. 2C). There was no observable increase in dead cells over the time course and concentrations of TNF-a and ionophore used in this study (FIG. 10A-C), suggesting PS externalization in these experiments was not due to cell death. Silencing of TMEM16E or TMEM16F had no effect on TNF-a-induced expression of TF on the cell surface (FIG. 2D). TMEM16 proteins can also function as ion channels and have been implicated in regulating intracellular Ca2+ flux in response to G-protein coupled receptor signaling (Yang et al., Cell 151 : 111-122 (2012); Yu et al., Elife 4: 1-23 (2015); Cabrita et al., FASEB J 31 :2123 -2134 (2017)). Therefore, TMEM16E or TMEM16F could affect PS externalization indirectly through regulation of Ca2+ transients. To test this possibility, intracellular Ca2+ was measured following stimulation with thrombin, which induces rapid Ca2+ elevation in ECs. Silencing of TMEM16E or TMEM16F did not significantly affect intracellular Ca2+ flux in ECs (FIG. 2E and FIG. 2F). These results suggest that TMEM16E and TMEM16F regulate EC procoagulant activity via PS externalization.
PS externalization during thrombotic injury occurs on the vessel wall
[00123] In order to directly observe the endothelial contribution of PS to thrombus formation in vivo, intravital microscopy was used to monitor PS, platelet, and fibrin accumulation in arterioles following laser injury to the vessel wall. The kinetics, localization, and area under the curve for PS was assessed by injecting fluorescently-conjugated annexin V. Platelets and fibrin were monitored with fluorescently conjugated anti-CD42b- and fibrin-specific antibodies, respectively. Following laser injury, PS externalization was consistently localized along the vessel wall, not in the growing platelet aggregate, and increased gradually, usually plateauing by 180 seconds (FIG. 3A-G and FIG. 11A-D) PS externalization consistently extended proximal and distal to the site of laser ablation and in vessels of smaller diameter, often spread to the opposite wall (FIG. 3A-G and FIG. 11A-D). Addition of the integrin a2bb3 (glycoprotein Ilb/IIIa) antagonist eptifibatide prevented platelet accumulation at the site of injury but had no effect on total PS extemalization at the site of injury (FIG. 3B-E). Annexin V was employed at a dose of 0.025 pg/g body weight, which is significantly lower than annexin V doses shown to inhibit thrombosis in other models (Thiagarajan and Benedict, Circulation 96:2339-2347 (1997); Rbmisch et al., Thromb Res 61 :93-104 (1991)). Annexin V at this dose did, however, result in a mild reduction in fibrin formation, with no effect on platelet accumulation (FIG. 12A-E). Use of other anionic phospholipid probes that bind PS such as lactadherin and pSIVA demonstrated an identical vessel wall pattern of PS exposure following laser injury (FIG. 3F and FIG. 3G).
Mice lacking TMEM16E have reduced fibrin formation following vessel injury
[00124] Because TMEM16E is an important player in EC procoagulant activity, knockout of TMEM16E in mice could alter thrombosis. Encoded by Ano5, TMEM16E is highly expressed in skeletal muscle. TMEM I 6E“ mice are overtly healthy but demonstrate defective muscle repair (Griffin et al., Hum Mol Genet 25: 1900-1911 (2016)). However, blood coagulation in TMEM16E" ' mice has not been studied. To determine whether TMEM16E plays a role in thrombus formation, platelet and fibrin accumulation were monitored by intravital microscopy following laser injury of the cremasteric arteriole. TMEM16E /_ mice demonstrated a small but significant decrease in fibrin formation (15.9%, median AUC 9.9 x 109 relative fluorescent units for TMEM16+ + and 8.3 x 109 for TMEM16E' ') and no difference in platelet accumulation compared to TMEM16E+ + littermate controls (Figs. 4A-4D and FIG. 41). To eliminate the contribution of platelet-mediated thrombosis and to clarify the contribution of platelet versus vessel wall PS, animals were treated with eptifibatide to prevent platelet accumulation. Under eptifibatide treatment, TMEM 16E" mice demonstrated a more significant reduction in fibrin formation (43.5%, median AUC 1.53 x 1010 relative fluorescent units [RFU] for TMEM16+/+ and 0.66 x 1010 for TMEM16E’7’) following laser injury compared to littermate controls (Figs. 4E-H and FIG. 4J). This suggested that platelet- mediated fibrin formation partially obscured TMEM16E-mediated thrombosis in animals not treated with eptifibatide. The diminished fibrin deposition in TMEM I 6E" mice could not be attributed to differences in baseline ex vivo coagulation parameters or platelet count (FIG. 13A- E). To assess whether TMEM16E was required for hemostasis, 8- to 10-week-old TMEM16E' ' and TMEM16E+ + littermate control mice were tested in a tail clip bleeding assay. TMEM16E" ' mice did not demonstrate excessive bleeding (Figs. 4K and 4L).
TMEM16 inhibitors reduce EC procoagulant activity
[00125] Ca2+ ionophore induced PS externalization on ECs in a dose-dependent manner (FIG. 5A). PS externalization alone did not promote factor Vila catalyzed factor Xa generation, presumably due to an absence of TF expression on the endothelial surface (FIG. 5B, gray bars). However, following stimulation with TNF-a, ionophore A23187 promoted a synergistic, dosedependent, increase in factor Vila catalyzed factor Xa generation (FIG. 5B, red bars). This augmentation of procoagulant activity was TF- and PS-dependent, as it was abolished with anti- TF antibody or lactadherin (FIG. 5B). Pharmacologic inhibition of TMEM16 was tested to investigate if it could reduce EC procoagulant activity. CaCCinh-AOl and benzbromarone are unrelated small molecules that have broad activity against TMEM16 channels including TMEM16E and TMEM16F (Zaitseva et al., Cell Host Microbe 22:99-110. e7 (2017); Centeio et al., IntJMol Set 21 :2557 (2020); Braga et al., Nature 594:88-93 (2021); Martins et al., Proc Natl Acad Sci USA 108: 18168-18172 (2011); Harper and Poole, Cell Death Dis 4: 1-5 (2013); Suzuki et al., J Biol Chem 288: 13305-13316 (2013)). Both of these drugs at 10 pM concentrations completely inhibit Ca2+-ionophore-stimulated PS exposure in HUVECs (FIG. 5C). CaCCinh-AOl and benzbromarone each inhibited factor Xa generation in a dose-dependent manner with an IC50 of 2.0 pM and 3.2 pM, respectively (FIG. 5D). Given the ability of TMEM16 antagonists to affect intracellular Ca2+ flux and therefore regulate PS externalization indirectly (Harper and Poole, Cell Death Dis 4:1-5 (2013)), CaCCinh-AOl and benzbromarone were tested to investigate whether reduced procoagulant activity is primarily via suppression of intracellular Ca2+ elevation. At 10 pM, neither CaCCinh-AOl nor benzbromarone inhibited intracellular Ca2+ flux in ECs, in contrast to the phospholipase C inhibitor U73122 which completely abolished intracellular Ca2+ elevation in response to thrombin (Figs. 5E and 5F). Benzbromarone did reduce Ca2+ transients at higher concentrations, however CaCCinh-AOl did not (FIG. 5F). Overall, these results suggest TMEM16 antagonism with benzbromarone and CaCCinh-AOl reduces procoagulant activity in ECs primarily by inhibiting PS externalization.
The TMEM16 inhibitor benzbromarone protects against thrombosis without increasing bleeding [00126] As there is extensive clinical experience using benzbromarone for the treatment of gout (Azevedo et a ., Adv Rheumatol (London, England) 59:37 (2019)), benzbromarone was tested to investigate whether it demonstrates antithrombotic properties in vivo. Wild-type C57BL/J6 mice were treated with intraperitoneal injection of benzbromarone (5 pg/g body weight) one hour prior to assessing thrombus formation following laser injury of the cremasteric vasculature. Benzbromarone reduced both platelet accumulation (55.1%, median AUC 6.28 x IO10 RFU for vehicle and 2.82 x IO10 for BBR) and fibrin formation (56.7%, median AUC 1.90 x 1010 RFU for vehicle and 0.85 x 1010 for BBR) following vessel injury (Figs. 6A-6D and 61). Mice were treated with eptifibatide to prevent platelet aggregation, and in the absence of platelet accumulation, benzbromarone still reduced fibrin formation (83.2%, median AUC 2.5 x 1010RFU for vehicle and 0.43 x 1010 for BBR) following laser ablation (Figs. 6E-6H and 6 J). Benzbromarone did not result in excessive bleeding following tail amputation (Figs. 6K and 6L).
Discussion
[00127] Biochemical and in vitro studies have established that a membrane surface containing anionic phospholipid, most notably PS, is essential to enable blood coagulation at physiologic reaction kinetics (Zwaal et al., Biophys Acta - Rev Biomembr 1376:433-453 (1998)). How PS externalization operates in vivo to support hemostasis and thrombosis is less well understood because exposure of PS is not captured by routine clinical measurements, and investigation of procoagulant PS has largely focused on platelets or other blood cell components. Our data suggest that the vessel wall is a major source of PS externalization to support thrombus formation. Previous mechanistic studies on PS externalization in blood coagulation have largely been limited to a single PLS, TMEM16F. Here, another PLS, TMEM16E, is identified as a novel positive regulator of PS externalization and procoagulant activity in ECs that may participate in thrombosis. TMEM16 inhibitors decreased EC procoagulant activity and protected against thrombosis without increased bleeding complications. These observations suggest new areas of investigation into endothelial membrane phospholipid dynamics that may uncover future therapeutic targets for pathologic blood clotting.
[00128] As cellular mediators of hemostasis and thrombosis, a proportion of activated platelets form membrane blebs containing externalized PS and support thrombin generation (Sims et al., J Biol Chem 264:17049-17057 (1989)). Other blood-contacting cells including monocytes (Del Conde et al., Arterioscler Thromb Vase Biol 25: 1065-1070 (2005)), erythrocytes (Wood et al., Blood 88:1873-1880 (1996); Bevers et al., Blood 79:380-388 (1992)), and ECs (Gao et al., PLoS One 2015;10: 1-16 (2015); Bombeli et al., Blood 89:2429-42 (1997); Popescu et al., Blood 116:993-1001 (2010)) can also support coagulation in a PS-dependent manner. The laser ablation model of vascular injury coupled with intravital microscopy has the ability to discern vessel wall- mediated versus platelet-mediated contributions to thrombosis (Higgins et al., J Clin Invest 128: 1471-1484 (2018); Jasuja et al., Blood 116:4665-4674 (2010); Vandendries et al., Proc Natl AcadSci 104:288-292 (2007); Kim et al., Blood 122: 1052-1061 (2013)). Employing three different PS probes, it is observed that following vessel injury, accessible PS is primarily is localized on the vessel wall, not platelets, despite the formation of large platelet aggregates (FIG. 3A-G and FIG. 11A-D). While unexpected, our results are consistent with prior studies demonstrating that coagulation complex assembly and fibrin generation occur along the vessel wall, and can be independent of platelet accumulation (Vandendries et al., Proc Natl Acad Sci 104:288-292 (2007); Kim et al., Blood 122: 1052-1061 (2013); Ivanciu et al., Blood 124: 1705-1714 (2014)). Both PS extemalization and fibrin formation are detected on the vessel wall despite inhibition of platelet accumulation with eptifibatide (FIG. 3A-G, FIG. 4A-L and FIG. 6A-L, and FIG. 11A-D). In summary, our data expands current models of thrombosis and supports a framework where the activated endothelium promotes thrombosis via PS extemalization.
[00129] The degree to which laser ablation results in endothelial disruption versus simply activation is not known, and these effects are likely to vary among injuries. Fibrin formation in this system occurs in pockets along the endothelium and subendothelium where PS and TF are likely to be exposed concurrently following laser injury. Similar to certain pharmacologic stimuli, laser pulse induces a rapid Ca2+ elevation in endothelial cells that propagates to cellular neighbors (Atkinson et al., Blood 116:4675-4683 (2010)), and likely explains the proximal, distal, and circumferential extension beyond the site of laser ablation (FIG. 3A-G and FIG. 11A-D). Annexin V sum intensity fluorescence did not correlate with the size of injury induced by laser ablation. This is in contrast to fibrin or platelet accumulation, which correlates well with injury size (FIG. 14A-C) (Grover et al., J Thromb Haemost 18:3078-3085 (2020)). The lack of correlation between annexin V sum intensity and laser-induced injury size is likely due to in part to limitations of quantifying annexin V in a single Z-plane. In smaller vessels, PS extemalization often extends to the opposite vessel wall and results in a larger measured sum fluorescent intensity independent of injury size (FIG. 3A-G and FIG. 11A-D). Despite these caveats, annexin V qualitative staining is very consistent, and the progression of PS externalization away from the site of injury occurs universally following laser ablation of all intensities.
[00130] Inflammatory stimuli such as lipopolysaccharide (Higgins et al., J Clin Invest 128: 1471-1484 (2018)) or TNF-a (this study) lead to concurrent expression of TF and externalization of PS, which together support procoagulant activity. Furthermore, TF-dependent factor Xa generation is augmented by increasing PS externalization (FIG. 5B). PS externalization in ECs, similar to other cells, including platelets, appears to result from TMEM16 activation via sustained elevations in intracellular Ca2+ (Bevers and Williamson, Physiol Rev 96:605-645 (2016)). Thus, TMEM16E and TMEM16F couple Ca2+ signaling to procoagulant activity in ECs. Some data suggest that TMEM16E and TMEM16F may affect Ca2+ flux itself (Cabrita et al., FASEB J 31 :2123-2134 (2017)), whereas our data in ECs are similar to studies of platelets and lymphocytes from patients with Scott syndrome, where TMEM16F deficiency abolished PS externalization without inhibiting intracellular Ca21 elevation (Munnix et al., Thromb Haemost 89:687-95 (2003); Kmit et al., Cell Death Dis 4: 1-8 (2013)). While apoptosis of ECs results in PS externalization, and therefore procoagulant activity (Bombeli et al., Blood 89:2429-42 (1997)), there was no increase in cell death over the time course and concentrations of TNF-a and ionophore used in this study (FIG. 2A-F and FIG. 10A-C). Clearly, viable cells can expose PS (Segawa et al., Proc Natl Acad Sci USA 108: 19246-19251 (2011)) and this exposure is reversible (Balasubramanian et al., J Biol Chem 282: 18357-18364 (2007); Gao et al., PLoS One 2015; 10: 1- 16 (2015)), as flippase activity of P4-ATPases restores lipid asymmetry ( Segawa et al., J Biol Chem 291 :762-772 (2016)). Our targeted screen identified the flippase ATP11C as a possible negative regulator of EC procoagulant activity (FIG. 1A). PS exposure on the endothelium may therefore be a mechanism by which both apoptotic or non-apoptotic inflammatory stimuli contribute to thrombosis. The caspase-activated PLS Xkr9 was also identified as a potential positive regulator of EC procoagulant activity (FIG. 1A). This finding may implicate other mechanisms of procoagulant PS externalization in ECs that may take on greater importance in distinct pathophysiologic states.
[00131] The TMEM16 family has 10 members, all of which act as Ca2+-activated ion channels, PLSs, or in certain cases, perhaps both (Whitlock and Hartzell, Anna Rev Physiol 79: 119-143 (2017)). The physiologic function of many family members is unknown. TMEM16A and TMEM16B are bona fide Ca2+-activated chloride channels, whereas structural and biochemical studies establish a distinct “scrambling” domain of TMEM16F and its closest paralog TMEM16E that allows for transmembrane phospholipid exchange (Whitlock et al., J Gen Physiol 2018;150: 1498-1509 (2018); Gyobu et al., Mol Cell Biol 36:645-659 (2016); Yu et al., Elife 4:1- 23 (2015); Foltz et al., J Cell Biol 220:e202007059 (2021); Feng et al., Cell Rep 28:567-579 (2019)). TMEM16F is ubiquitously expressed whereas TMEM16E exhibits tissue-restricted expression. High amounts of TMEM16E are found in skeletal muscle and most functional studies of TMEM16E have focused on muscle physiology. TMEM16E regulates muscle regeneration, myoblast fusion and myocyte membrane repair (Whitlock et al., J Gen Physiol 2018; 150: 1498- 1509 (2018); Griffin et al., Hum Mol Genet 25: 1900-1911 (2016); Sui et al., Cell Death Dis 9:609 (2018)). Curiously, one function of TMEM16E, membrane repair following injury, appears to be independent of scramblase domain function (Foltz et al., J Cell Biol 220:e202007059 (2021)). Mutations in TMEM16E cause limb girdle muscular dystrophy type 2L (LGMD2L) (Bolduc et al., Am J Hum Genet 86:213-221 (2010)), an autosomal recessive, late-onset muscular dystrophy, or gnathodiaphyseal dysplasia (GDD) (Tsutsumi et al., Am J Hum Genet 74: 1255-1261 (2004)), an extremely rare autosomal dominant pediatric bone disease. Neither of these conditions have been associated with abnormal hemostasis or thrombosis, but this possibility has not been investigated. [00132] A series of investigations has demonstrated that TMEM16E readily scrambles phospholipid, but the localization of TMEM16E within cells and importance of TMEM16E lipid scrambling per se, is unclear (Whitlock et al., Gen Physiol 2018;150:1498-1509 (2018); Gyobu et al., Mol Cell Biol 36:645-659 (2016)). Some studies suggest TMEM16E is predominantly expressed in intracellular compartments, such as the ER (Gyobu et al., Mol Cell Biol 36:645-659 (2016); Mizuta et al., Biochem Biophys Res Commun 357:126-132 (2007); Duran et al., Am J Physiol - Cell Physiol 302:482-493 (2012)), whereas others suggest it may also be located on the plasma membrane (Whitlock et al., J Gen Physiol 2018;150: 1498-1509 (2018); Di Zanni et al., Cell Mol Life Sci 75: 1657-1670 (2018)). Of relevance to our own thrombosis model, one study in myocytes found that laser injury induced redistribution of TMEM16E to the plasma membrane within seconds (Foltz et al., J Cell Biol 220:e202007059 (2021)). The mechanism by which TMEM16E regulates PS extemalization on the plasma membrane is therefore unclear (Whitlock et al., J Gen Physiol 2018;150: 1498-1509 (2018)). Furthermore, it is unknown why certain cells such as myocytes and ECs require TMEM16E for PS extemalization when they also express high levels of TMEM16F (Whitlock et a\., J Gen Physiol 2018;150: 1498-1509 (2018)). TMEM16E- null animals do not compensate by increasing expression of TMEM16F, for example (Foltz et al., JCellBiol 220:e202007059 (2021); Xu et al., Skelet Muscle 5: 1-14 (2015)). Dual silencing ofboth TMEM16E and TMEM16F did not result in additive reduction in procoagulant activity, likely because silencing of either TMEM16E or TMEM16F nearly completely inhibited PS extemalization (FIG. 1A-G and FIG. 2A-F). This result raises the possibility that TMEM16E and TMEM16F are epistatic to one another, regulating PS extemalization through direct interaction or through a linear pathway involving both proteins, perhaps involving ER-plasma membrane contact sites which control lipid trafficking (Chung et al., Science 349:428-432 (2015)).
[00133] Prior studies have evaluated the role of TMEM16F in thrombosis but have focused on platelet-based models. Global TMEM16F-null mice demonstrated delayed occlusion in a ferric chloride carotid injury model of thrombosis and a mild bleeding diathesis (Yang et al., Cell 151 : 111-122 (2012)). Animals with a platelet-specific conditional TMEM16F deletion exhibited reduced platelet aggregates in a reactive oxygen species-based model of mesenteric venule thrombosis (Fujii et al., Proc Natl Acad Sci USA 112:12800-12805 (2015)) and were protected from ferric chloride carotid thrombosis but not from thrombo-inflammatory ischemic stroke (Baig et al., Arterioscler Thromb Vase Biol 36:2152-2157 (2016)). In contrast, platelet-specific deletion of phosphatidylinositol transfer protein a (PITPa) abolished PS extemalization in platelets due to a lack of IP3-induced Ca2+ elevation but had no effect on thrombosis in a ferric chloride model (Zhao et al., Nat Commun 8: 1-11 (2017)). These studies support the possibility that sources beyond the platelet, such as the endothelium and regulators other than TMEM16F control PS extemalization in hemostasis and thrombosis.
[00134] TMEM16 inhibition with two unrelated compounds reduced endothelial PS extemalization and procoagulant activity. One such compound, the uricosuric agent benzbromarone, has been used for decades to treat gout worldwide (Perez-Ruiz et al., Ann Rheum Dis 57:545-549 (1998); Heel et al., Drugs 14:349-66 (1977)). Thus, TMEM16 inhibition could be further explored as an antithrombotic strategy, perhaps one targeting Ca2+-dependent PS extemalization during inflammation. Benzbromarone is generally considered safe but was not approved by the FDA due to serious but exceedingly rare hepatotoxicity events estimated at 1 in 17,000 (Azevedo et al., Adv Rheumatol (London, England) 59:37 (2019)). Time-limited treatment with benzbromarone for inhibition of prothrombotic PS extern al izati on may have a more tolerable safety profile.
[00135] In summary, the work presented here establishes TMEM16 proteins as drivers of EC procoagulant activity. Two PLSs are identified, TMEM16F, known to contribute to procoagulant activity in platelets, and TMEM16E, which has not previously been implicated in coagulation. The vessel wall likely contributes procoagulant phospholipid to support thrombosis. Moreover, fibrin generation, the terminal product in coagulation activation, is impaired in TMEM16E-deficient mice following vascular injury. TMEM16 antagonism likely dampens prothrombotic endothelium and inhibits thrombosis without increasing bleeding complications. This study should foster future investigation into the significance of endothelial lipid scrambling in thrombotic disease and the role of TMEM16 inhibition as a viable antithrombotic target.
SEQUENCE LISTING
<110> Beth Israel Deaconess Medical Center
<120> COMPOSITIONS AND METHODS FOR REGULATING PROCOAGULANT ACTIVITY AND THROMBOSIS
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Claims

WHAT IS CLAIMED IS
1. A method of treating a thrombotic disorder, comprising administering a therapeutically effective amount of one or more TMEM16 inhibitors to a patient in need thereof.
2. The method of claim 1, wherein the TMEM16 is TMEM16E, TMEM16F, or any combination thereof.
3. The method of claim 2, wherein the one or more TMEM16 inhibitors is benzbromarone, isoquercetin, ivermectin, crofelemer, nitazoxanide, hexachlorophene, dichlorophen, nitazoxanide, trifluoperazine, or a combination thereof.
4. The method of claim 3, wherein the one or more TMEM16 inhibitors comprises benzbromarone.
5. The method of claim 4, wherein the one or more TMEM16 inhibitors further comprises isoquercetin.
6. The method of claim 5, wherein the one or more TMEM16 inhibitors comprise benzbromarone and isoquercetin and wherein administering results in a synergistic antithrombotic effect.
7. The method of claim 3, further comprising administering zafirlukast.
8. The method of claim 7, wherein administering the one or more TMEM16 inhibitors further comprising administering zafirlukast results in a synergistic antithrombotic effect.
9. The method of claim 4, wherein a therapeutically effective amount of benzbromarone is from about 50 mg to about 800 mg per day.
10. The method of claim 4, wherein a therapeutically effective plasma concentration of benzbromarone is from about 1 to about 30 micromolar.
11. The method of claim 4, wherein a therapeutically effective amount of benzbromarone is about 5 pg/g.
12. The method of claim 9, wherein the therapeutically effective amount of benzbromarone is administered between about every 12 to about every 24 hours.
13. The method of claim 9, wherein the therapeutically effective amount of benzbromarone is administered about every 24 hours.
14. The method of claim 1, wherein the thrombotic disorder is a thrombotic disease, hypercoagulation, blood coagulation, TMEM16-induced coagulation, coronary artery disease, cardiovascular disease, procoagulant endothelial cell dysfunction, procoagulant pulmonary epithelial cell dysfunction, aberrant procoagulant activity, pathological phosphoserine extemalization, dysregulated blood coagulation, antiphospholipid antibody syndrome, SARS- CoV-2 pneumonia, thrombosis during severe SARS-CoV-2 infection, post-acute sequelae of SARS CoV-2 infection, cancer-associated thrombosis, refractory thrombosis, myocardial infarction, stroke, venous thromboembolism, disseminated intravascular coagulation (DIC), sepsis, paroxysmal nocturnal hemoglobinuria, antiphospholipid antibody syndrome, myeloproliferative disorders, inherited red cell disorders associated with thrombosis, sickle cell disease, hereditary xerocytosis, acute coronary syndrome, atrial fibrillation, or any combination thereof.
15. The method of claim 1, wherein administering the therapeutically effective amount of one or more TMEM16 inhibitors does not increase bleeding complications, bleeding time, bleeding risk, or any combination thereof.
16. The method of claim 1, wherein administering the therapeutically effective amount of one or more TMEM16 inhibitors results in a reduction in at least one indicator of thrombotic disease, results in at least one indicator of thrombotic disease remaining within an acceptable range, or a combination thereof.
17. The method of claim 16, wherein the at least one indicator of thrombotic disease is D-dimer, thrombin antithrombin complex, prothrombin activation peptide Fl +2, procoagulant extracellular vesicle TF cofactor activity, factor Xa generation, thrombin generation, or plasma endogenous thrombin potential.
18. The method of claim 1, wherein administering the therapeutically effective amount of one or more TMEM16 inhibitors results in concentrations of D-dimer in the patient remaining below about 500 ng/mL.
19. The method of claim 1, wherein the one or more TMEM16 inhibitors is administered in combination with a blood-thinning medication, chemotherapy, or a drug indicated for the treatment of heart disease.
20. The method of claim 19, wherein the one or more TMEM16 inhibitors is administered in combination with warfarin, heparin, low molecular weight heparins, ultra-low molecular weight heparins, direct oral anticoagulants targeting thrombin, factor Xa, factor Xia, parenteral direct thrombin inhibitors, argatroban, aspirin, P2Y12 inhibitors, clopidogrel, or a combination thereof.
21. The method of claim 1, wherein the thrombotic disorder is associated with COVID-19.
22. The method of claim 1, wherein the patient has cancer.
23. The method of claim 22, wherein the cancer is multiple myeloma, hematologic cancer, adenocarcinoma, cancer of the pancreas, stomach, ovaries, prostate, colon, lung, brain, breast, kidney, skin, cervix, or ear-nose-throat cancer.
24. A method for treating hypercoagulation related to COVID- 19, comprising administering a therapeutically effective amount of one or more TMEM16 inhibitors to a patient in need thereof.
25. The method of claim 24, wherein the COVID- 19 is post-acute sequelae of SARS CoV-2 infection.
26. The method of claim 24, wherein the TMEM16 is TMEM16E, TMEM16F, or any combination thereof.
27. The method of claim 26, wherein the one or more TMEM16 inhibitors is benzbromarone, isoquercetin, ivermectin, crofelemer, nitazoxanide, hexachlorophene, dichlorophen, nitazoxanide, trifluoperazine, or a combination thereof.
28. The method of claim 27, wherein the one or more TMEM16 inhibitors comprises benzbromarone.
29. The method of claim 28, wherein the one or more TMEM16 inhibitors further comprises isoquercetin.
30. The method of claim 29, wherein administering the one or more TMEM16 inhibitors comprising benzbromarone and isoquercetin results in a synergistic anti-hypercoagulation effect.
31. The method of claim 30, wherein a therapeutically effective amount of benzbromarone is from about 50 mg to about 800 mg per day.
32. The method of claim 30, wherein a therapeutically effective plasma concentration of benzbromarone is from about 1 to about 30 micromolar.
33. The method of claim 30, wherein a therapeutically effective amount of benzbromarone is about 5 pg/g.
34. The method of claim 31, wherein the therapeutically effective amount of benzbromarone is administered between about every 12 to about every 24 hours.
35. The method of claim 31, wherein the therapeutically effective amount of benzbromarone is administered about every 24 hours.
36. The method of claim 24, wherein the hypercoagulation related to COVLD-19 is microcoagulation, microvascular thrombosis, immunothrombosis, TMEM16-induced coagulation, coronary artery disease, cardiovascular disease, procoagulant endothelial cell dysfunction, procoagulant pulmonary epithelial cell dysfunction, aberrant procoagulant activity, pathological phosphoserine externalization, dysregulated blood coagulation, antiphospholipid antibody syndrome, SARS-CoV-2 pneumonia, thrombosis during severe SARS-CoV-2 infection, refractory thrombosis, myocardial infarction, stroke, venous thromboembolism, disseminated intravascular coagulation (DIC), sepsis, paroxysmal nocturnal hemoglobinuria, antiphospholipid antibody syndrome, myeloproliferative disorders, acute coronary syndrome, atrial fibrillation, or any combination thereof.
37. The method of claim 24, wherein administering the therapeutically effective amount of one or more TMEM16 inhibitors does not increase bleeding complications, bleeding time, bleeding risk, or any combination thereof.
38. The method of claim 24, wherein administering the therapeutically effective amount of one or more TMEM16 inhibitors results in a reduction in at least one indicator of hypercoagulation, results in at least one indicator of hypercoagulation remaining within an acceptable range, or a combination thereof.
39. The method of claim 38, wherein the at least one indicator of hypercoagulation is D-dimer, thrombin antithrombin complex, prothrombin activation peptide Fl +2, procoagulant extracellular vesicle TF cofactor activity, factor Xa generation, thrombin generation, or plasma endogenous thrombin potential.
40. The method of claim 24, wherein administering the therapeutically effective amount of one or more TMEM16 inhibitors results in concentrations of D-dimer in the patient remaining below about 500 ng/mL.
41. The method of claim 24, wherein the one or more TMEM16 inhibitors is administered in combination with oral antiviral drugs, intravenous antiviral drugs, monoclonal antibodies, bloodthinning medications, or a drug indicated for the treatment of heart disease.
42. The method of claim 41, wherein the one or more TMEM16 inhibitors is administered in combination with direct oral anticoagulants, dabigatran, rivaroxaban, apixaban, edoxaban, betrixaban, Paxlovid, molnupiravir, remdesivir, Bebtelovimab, Evusheld, warfarin, heparin, low molecular weight heparins, ultra-low molecular weight heparins, direct oral anticoagulants targeting thrombin, factor Xa, factor Xia, parenteral direct thrombin inhibitors, argatroban, aspirin, P2Y12 inhibitors, clopidogrel, or a combination thereof.
43. The method of claim 27, further comprising administering zafirlukast.
44. The method of claim 43, wherein administering the one or more TMEM16 inhibitors further comprising administering zafirlukast results in a synergistic anti-hypercoagulation effect.
45. A method for reducing cancer-associated thrombosis, comprising administering to a patient a therapeutically effective amount of one or more TMEM16 inhibitors.
46. The method of claim 45, wherein the TMEM16 is TMEM16E, TMEM16F, or any combination thereof.
47. The method of claim 45, wherein the one or more TMEM16 inhibitors is benzbromarone, isoquercetin, ivermectin, crofelemer, nitazoxanide, hexachlorophene, dichlorophen, nitazoxanide, trifluoperazine, or a combination thereof.
48. The method of claim 47, wherein the one or more TMEM16 inhibitors comprises benzbromarone.
49. The method of claim 48, wherein the one or more TMEM16 inhibitors further comprises isoquercetin.
50. The method of claim 49, wherein administering the one or more TMEM16 inhibitors comprising benzbromarone and isoquercetin results in a synergistic antithrombotic effect.
51. The method of claim 47, further comprising administering zafirlukast.
52. The method of claim 51, wherein administering the one or more TMEM16 inhibitors further comprising administering zafirlukast results in a synergistic antithrombotic effect.
53. The method of claim 48, wherein a therapeutically effective amount of benzbromarone is from about 50 mg to about 800 mg per day.
54. The method of claim 48, wherein a therapeutically effective plasma concentration of benzbromarone is from about 1 to about 30 micromolar.
55. The method of claim 48, wherein a therapeutically effective amount of benzbromarone is about 5 pg/g.
56. The method of claim 53, wherein the therapeutically effective amount of benzbromarone is administered between about every 12 to about every 24 hours.
57. The method of claim 56, wherein the therapeutically effective amount of benzbromarone is administered about every 24 hours.
58. The method of claim 45, wherein the patient has a thrombotic disease, cardiovascular disease, procoagulant endothelial cell dysfunction, procoagulant pulmonary epithelial cell dysfunction, aberrant procoagulant activity, pathological phosphoserine externalization, dysregulated blood coagulation, antiphospholipid antibody syndrome, cancer-associated thrombosis, refractory thrombosis, myocardial infarction, stroke, venous thromboembolism, disseminated intravascular coagulation (DIC), sepsis, paroxysmal nocturnal hemoglobinuria, antiphospholipid antibody syndrome, myeloproliferative disorders, inherited red cell disorders associated with thrombosis, sickle cell disease, hereditary xerocytosis, acute coronary syndrome, atrial fibrillation, or any combination thereof.
59. The method of claim 45, wherein administering the therapeutically effective amount of one or more TMEM16 inhibitors does not increase bleeding complications, bleeding time, bleeding risk, or any combination thereof.
60. The method of claim 45, wherein administering the therapeutically effective amount of one or more TMEM16 inhibitors results in a reduction in at least one indicator of thrombotic disease, results in at least one indicator of thrombotic disease remaining within an acceptable range, or a combination thereof.
61. The method of claim 60, wherein the at least one indicator of thrombotic disease is D-dimer, thrombin antithrombin complex, prothrombin activation peptide Fl +2, procoagulant extracellular vesicle TF cofactor activity, factor Xa generation, thrombin generation, or plasma endogenous thrombin potential.
62. The method of claim 45, wherein administering the therapeutically effective amount of one or more TMEM16 inhibitors results in concentrations of D-dimer in the patient remaining below about 500 ng/mL.
63. The method of claim 45, wherein the one or more TMEM16 inhibitors is administered in combination with a blood-thinning medication, chemotherapy, or a drug indicated for the treatment of heart disease.
64. The method of claim 63, wherein the one or more TMEM16 inhibitors is administered in combination with warfarin, heparin, low molecular weight heparins, ultra-low molecular weight heparins, direct oral anticoagulants targeting thrombin, factor Xa, factor Xia, parenteral direct thrombin inhibitors, argatroban, aspirin, P2Y12 inhibitors, clopidogrel, or a combination thereof.
65. The method of claim 45, wherein the cancer is multiple myeloma, hematologic cancer, adenocarcinoma, cancer of the pancreas, stomach, ovaries, prostate, colon, lung, brain, breast, kidney, skin, cervix, or ear-nose-throat cancer.
66. A therapeutic composition suitable to treat a thrombotic disorder, comprising a therapeutically effective amount of one or more TMEM16 inhibitors, and pharmaceutically acceptable excipients.
67. The therapeutic composition of claim 66, wherein the TMEM16 is TMEM16E, TMEM16F, or any combination thereof.
68. The therapeutic composition of claim 66, wherein the one or more TMEM16 inhibitors is benzbromarone, isoquercetin, ivermectin, crofelemer, nitazoxanide, hexachlorophene, dichlorophen, nitazoxanide, trifluoperazine, or a combination thereof.
69. The therapeutic composition of claim 68, wherein the one or more TMEM16 inhibitors comprises benzbromarone.
70. The therapeutic composition of claim 69, wherein the one or more TMEM16 inhibitors further comprises isoquercetin.
71. The therapeutic composition of claim 70, wherein administering the one or more TMEM16 inhibitors comprising benzbromarone and isoquercetin results in a synergistic antithrombotic effect.
72. The therapeutic composition of claim 68, further comprising administering zafirlukast.
73. The therapeutic composition of claim 72, wherein administering the one or more TMEM16 inhibitors further comprising administering zafirlukast results in a synergistic antithrombotic effect.
74. The therapeutic composition of claim 69, wherein a therapeutically effective amount of benzbromarone is from about 50 mg to about 800 mg per day.
75. The therapeutic composition of claim 69, wherein a therapeutically effective plasma concentration of benzbromarone is from about 1 to about 30 micromolar.
76. The therapeutic composition of claim 69, wherein a therapeutically effective amount of benzbromarone is about 5 pg/g.
77. The therapeutic composition of claim 69, wherein the therapeutically effective amount of benzbromarone is configured for administration between about every 12 to about every 24 hours.
78. The therapeutic composition of claim 77, wherein the therapeutically effective amount of benzbromarone is configured for administration between about every 24 hours.
79. The therapeutic composition of claim 66, wherein the thrombotic disorder is a thrombotic disease, cardiovascular disease, procoagulant endothelial cell dysfunction, procoagulant pulmonary epithelial cell dysfunction, aberrant procoagulant activity, pathological phosphoserine extemalization, dysregulated blood coagulation, antiphospholipid antibody syndrome, SARS- CoV-2 pneumonia, thrombosis during severe SARS-CoV-2 infection, post-acute sequelae of SARS CoV-2 infection, cancer-associated thrombosis, refractory thrombosis, myocardial infarction, stroke, venous thromboembolism, disseminated intravascular coagulation (DIC), sepsis, paroxysmal nocturnal hemoglobinuria, antiphospholipid antibody syndrome, myeloproliferative disorders, inherited red cell disorders associated with thrombosis, sickle cell disease, hereditary xerocytosis, acute coronary syndrome, atrial fibrillation, or any combination thereof.
80. The therapeutic composition of claim 66, wherein the therapeutically effective amount of one or more TMEM16 inhibitors does not increase bleeding complications, bleeding time, bleeding risk, or any combination thereof.
81. The therapeutic composition of claim 66, wherein the therapeutically effective amount of one or more TMEM16 inhibitors results in a reduction in at least one indicator of thrombotic disease, results in at least one indicator of thrombotic disease remaining within an acceptable range, or a combination thereof.
82. The therapeutic composition of claim 81, wherein the at least one indicator of thrombotic disease is D-dimer, thrombin antithrombin complex, prothrombin activation peptide Fl+2, procoagulant extracellular vesicle TF cofactor activity, factor Xa generation, thrombin generation, or plasma endogenous thrombin potential.
83. The therapeutic composition of claim 66, wherein the therapeutically effective amount of one or more TMEM16 inhibitors results in concentrations of D-dimer in a patient remaining below about 500 ng/mL.
EP23904458.9A 2022-12-12 2023-12-12 Compositions and methods for regulating procoagulant activity and thrombosis Pending EP4633622A2 (en)

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