WO2019071171A1 - Procédé de traitement de la drépanocytose - Google Patents

Procédé de traitement de la drépanocytose Download PDF

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Publication number
WO2019071171A1
WO2019071171A1 PCT/US2018/054676 US2018054676W WO2019071171A1 WO 2019071171 A1 WO2019071171 A1 WO 2019071171A1 US 2018054676 W US2018054676 W US 2018054676W WO 2019071171 A1 WO2019071171 A1 WO 2019071171A1
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arq
amount
pharmaceutical formulation
wherien
scd
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PCT/US2018/054676
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English (en)
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Jaehyung Cho
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The Board Of Trustees Of The University Of Illinois
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Publication of WO2019071171A1 publication Critical patent/WO2019071171A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/4353—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems
    • A61K31/437—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems the heterocyclic ring system containing a five-membered ring having nitrogen as a ring hetero atom, e.g. indolizine, beta-carboline
    • 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
    • A61K31/16—Amides, e.g. hydroxamic acids
    • A61K31/17—Amides, e.g. hydroxamic acids having the group >N—C(O)—N< or >N—C(S)—N<, e.g. urea, thiourea, carmustine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • 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

Definitions

  • Sickle cell disease can refer to a group of inherited red blood cell disorders.
  • the red blood cells can become hard and sticky and look like a C-shaped farm tool called a "sickle.”
  • SCD results in a constant shortage of red blood cells and can cause additional problems due to sickle red blood cell-induced clogs in the blood vessels.
  • SCD results in significant morbidity and mortality.
  • the only cure for SCD is a bone marrow or stem cell transplant.
  • Current SCD therapies include symptom management and treatment with hydroxyurea (HU).
  • HU hydroxyurea
  • SCD patients receiving HU therapy can suffer from vaso- occlusive crises. As such, there exists a need for improved treatments for SCD.
  • the method can further include administering an amount of hydroxyurea (HU) to the subject in need thereof.
  • the amount of ARQ 092 and the amount of HU can be administered at about the same time.
  • the amount of ARQ 092 and the amount of HU can be administered at different times.
  • the amount of HU can be administered before the amount of ARQ 092.
  • the amount of ARQ 092 can be contained in a pharmaceutical formulation.
  • the amount of HU is contained in a pharmaceutical formulation.
  • the amount of ARQ 092 and the amount of HU can be contained in the same pharmaceutical formulation.
  • the amount of ARQ 092 and the amount of HU are contained in separate pharmaceutical formulations.
  • the amount of ARQ 092 can be an effective amount.
  • the effective amount of ARQ 092 can range from about 0.01 mg/kg body weight to about 25 mg/kg body weight.
  • the amount of HU can be an effective amount.
  • the effective amount of HU can range from about 10 mg/kg body weight to about 50 mg/kg body weight.
  • kits can include an amount of ARQ 092 or a pharmceutical formuation thereof; and instrcutions or indications for use contained in a tangible medium of expression, wherien the instructions or indications for use provide instructions for treating a sickle cell disease in a subject in need thereof by administering the amount of ARQ 092 or the pharmaceutical formulation thereof.
  • the kit can further include an amount of hydroxyurea or a pharmaceutical formulation thereof, and wherein the instructions or indications for use contained in a tangible medium of expression further provide instructions for treating a sickle cell disease in a subject in need thereof by administering the amount of HU or the pharmaceutical formulation thereof.
  • the instructions or indications for use contained in a tangible medium of expression can further provide instructions for treating a sickle cell disease in a subject in need thereof by administering the amount of HU or the pharmaceutical formulation thereof at about the same time as administering the amount of ARQ 092 or the pharmaceutical formulation thereof.
  • the instructions or indications for use contained in a tangible medium of expression can further provide instructions for treating a sickle cell disease in a subject in need thereof by administering the amount of HU or the pharmaceutical formulation thereof at a different time as administering the amount of ARQ 092 or the pharmaceutical formulation thereof.
  • the instructions or indications for use contained in a tangible medium of expression can further provide instructions for treating a sickle cell disease in a subject in need thereof by administering the amount of HU or the pharmaceutical formulation thereof prior to administering the amount of ARQ 092 or the pharmaceutical formulation thereof.
  • the amount of ARQ 092 can be an effective amount.
  • the effective amount of ARQ 092 can range from about 0.01 mg/kg body weight to about 250 mg/kg body weight.
  • the amount of HU can be an effective amount.
  • the effective amount of HU can range from about 10 mg/kg body weight to about 50 mg/kg body weight.
  • compositions can include an effective amount of ARQ 092, wherien the effective amount of ARQ 092 can effetive to treat a sickle cell disease or a symptom thereof in a subject in need thereof; and a pharmaceutically acceptable carrier.
  • the pharmaceutical formulation can further an amount of hydroxyurea (HU).
  • the effective amount of HU can be effetive to treat a sickle cell disease or a symptom thereof in a subject in need thereof.
  • the effective amount of ARQ 092 can beombive to reduce or mitigate one or more side effects of HU in the subject in need thereof.
  • the effective amount of ARQ 092 can range from about 0.01 mg/kg body weight to about 250 mg/kg body weight.
  • the effective amount of HU can range from about 10 mg/kg body weight to about 50 mg/kg body weight.
  • ARQ 092 for use in the treatmet of sickle cell disease.
  • descibred herien is ARQ 092 in combination with hydroxyurea for use in the treatment of sickle cell disease.
  • ARQ 092 for use in the prepartion of a medicament for the treatmet of sickle cell disease.
  • ARQ 092 in combination with hydroxy urea for use in the prepartion of a medicament for the treatmet of sickle cell disease.
  • FIGS. 1A-1J can demonstrate that ARQ 092 inhibits activation of neutrophils and platelets isolated from SCD patients in vitro.
  • Neutrophils FIGS. 1 A-1 D
  • platelets FIGS. 1 E- 1J
  • FIGS. 1A-1J can demonstrate that ARQ 092 inhibits activation of neutrophils and platelets isolated from SCD patients in vitro.
  • Neutrophils FIGS. 1 A-1 D
  • platelets FIGS. 1 E- 1J
  • FIG. 1 B-1 D Flow cytometry was performed using PE-conjugated control IgG or antibodies against total (ICRF44) or activated ⁇ 2 (CBRM1/5), or DyLight 488-conjugated FG. The geometric mean fluorescence intensity of antibodies was normalized to that of control IgG, and data are presented as fold increase compared with vehicle-treated, unstimulated cells.
  • FIG. 1 F Flow cytometry was performed to measure P-selectin exposure.
  • FIGGS. 1 G-H Platelets were stimulated with (FIG.
  • FIG. 11 Platelet agglutination was induced by 0.5 ⁇ g/ml vWF and 0.1 mg/ml ristocetin.
  • FIGS. 2A-2C can demonstrate that ARQ 092 perturbs heterotypic aggregation of neutrophils and platelets from SCD patients under stirring conditions.
  • Neutrophils and platelets isolated from SCD patients were pretreated with vehicle (0.1 % DMSO (-)) or 500 nM ARQ 092 and then incubated with FITC-conjugated anti-L-selectin and APC-conjugated anti-CD41 a antibodies, respectively.
  • Thrombin-activated platelets were mixed with neutrophils under stirring conditions, followed by flow cytometric analysis. Flow cytometry results are shown in FIG. 2A.
  • R1 leukocyte-platelet aggregates
  • FIG. 2B Cell-cell aggregation was measured by the number of cell-cell aggregates (R3).
  • FIGS. 3A-3F can demonstrate that oral administration of ARQ 092 blocks AKT phosphorylation and activation of neutrophils and platelets isolated from SCD mice ex vivo.
  • Vehicle (0.01 M phosphoric acid) or ARQ 092, 100 mg/kg, was given orally to SCD mice. Blood and bone marrow were collected 30 minutes after treatment.
  • Isolated neutrophils (FIGS. 3A-3C) or platelets (FIGS. 3D-3F) were incubated with or without 10 ⁇ fMLP or 0.025 U/ml thrombin for 2 minutes, respectively. (FIGS.
  • FIGS. 4A-44M can demonstrate that oral administration of HU and ARQ 092 has numerous beneficial effects in TNF-a-challenged SCD mice.
  • Intravital microscopy was performed as described in Methods. Neutrophils and platelets were labeled by infusion of Alexa Fluor 647-conjugated anti-Ly-6G and DyLight 488-conjugated anti-CD42c antibodies, respectively.
  • FIG. 4A Timeline for each treatment and surgery for intravital microscopy (IVM) in SCD mice. S: saline and PA: 0.01 M phosphoric acid.
  • FIG. 4B Representative images at various time points. The time "0" was set as the image capture was initiated for each vessel.
  • FIG. 4E The integrated median fluorescence intensities of anti-CD42c antibodies (F platelets) were plotted over time.
  • FIG. 4L Representative images.
  • FIGS. 5A-5F can demonstrate that co-administration of HU and ARQ 092 efficiently inhibits ⁇ 2 integrin function and ROS generation in stimulated neutrophils isolated from SCD mice ex vivo.
  • Vehicle (0.01 M phosphoric acid, -), HU, ARQ 092, or both inhibitors were given orally to TNF-a-challenged SCD mice as described in FIG. 4A.
  • Bone marrow was collected at 3 hours after TNF-a injection. Isolated neutrophils were incubated with or without fMLP.
  • FIGS. 5A-5B The surface level of ⁇ 2 integrin and soluble FG binding were measured by flow cytometry.
  • FIGS. 6A-6I can demonstrate that co-administration of HU and ARQ 092 efficiently inhibits numerous functions of activated platelets isolated from SCD mice ex vivo.
  • Vehicle (0.01 M phosphoric acid, -) or inhibitors were given orally to TNF-a-challenged SCD mice as described in FIGS. 5A-5F.
  • Blood was collected 3 hours after TNF-a injection. Isolated platelets were treated with or without thrombin.
  • FIG. 5A Flow cytometry was performed to measure P-selectin exposure.
  • FIGS. 5B-5D Platelets were pretreated with Ca 2+ dye and incubated with thrombin for 5 minutes and 2 mM CaC was then added. Ca 2+ release (FIG.
  • FIG. 5C Platelet agglutination was induced by 10 ⁇ g/ml vWF and 10 ⁇ g/ml botrocetin.
  • FIG. 5F Platelet aggregation was induced by thrombin. The representative agglutination or aggregation trace was obtained from three independent experiments.
  • FIG. 5G Platelets were incubated with DCFH-DA prior to thrombin stimulation. Intracellular ROS generation was measured by the DCF signal in flow cytometry.
  • FIGS. 7A-7B can demonstrate that HU reduces neutrophil and platelet activation via NO production.
  • SCD mice were pretreated by iv injection of PTIO, an NO scavenger (1 mg/kg) 30 minutes prior to TNF-a challenge.
  • Vehicle (0.01 M phosphoric acid, -) or inhibitors were given orally to TNF-a-challenged SCD mice as described in FIGS. 5A-5D.
  • Blood and bone marrow were collected 3 hours after TNF-a injection.
  • Isolated neutrophils (FIGS. 7A-7B) and platelets (FIG. 7C) were treated with or without fMLP (for 10 minutes) or thrombin (for 5 minutes), respectively.
  • Flow cytometry was performed to measure (FIGS.
  • FIGS. 8A-8B can demonstrate chimerism of SCD mice.
  • FIG. 8A Genotyping of genomic DNA isolated from mouse blood. T: SCD ( ⁇ - ⁇ -/-) mice, C: control hemozygous (Hbp+/-) mice, and W: C57BL/6 mice.
  • FIG. 8B Chimerism of SCD mice by Hb electrophoresis. Hb lysates, 2 ⁇ g, were electrophoresed on an acidic polyacrylamide gel containing urea. Human Hb (H), WT mouse (C57BL/6, M), and different SCD mice Hb. No mouse Hba was detected in SCD mice tested.
  • FIGS. 9A-9D can demonstrate the kinetics of AKT phosphorylation in neutrophils and platelets following agonist stimulation.
  • Neutrophils FIGS. 9A and 9C
  • platelets FIGS. 9B and 9D
  • Neutrophils or platelets were isolated from SCD patients (FIGS. 9A and 9B) and mice (FIGS. 9C and 9D).
  • Neutrophils or platelets were treated with or without fMLP (0.5 ⁇ for human or 10 ⁇ for mouse) or 0.025 U/ml thrombin, respectively, for 1 , 2, 5, or 10 minutes.
  • Equal amounts 50 ⁇ g) of cell lysate protein were immunoblotted. Representative blots were obtained from three independent experiments.
  • FIGS. 1 1A-1 1C can demonstrate an effect of anti-L-selectin and anti-CD41 a antibodies on neutrophil- platelet aggregation.
  • FIGS. 12A-12B can demonstrate inhibitory effects of ARQ 092 on AKT phosphorylation in SCD mouse neutrophils and platelets in vitro.
  • Neutrophils (FIG. 12A) and platelets (FIG. 12B) isolated from SCD mice were pre-treated with 0.1 % DMSO (0), or 5, 50, or 500 nM ARQ 092, followed by stimulation with 10 ⁇ fMLP or 0.025 U/ml thrombin. Equal amounts (50 ⁇ g) of cell lysate protein were immunoblotted. Representative blots were obtained from three independent experiments.
  • FIGS. 13A-13B can demonstrate inhibitory effects of ARQ 092 on AKT phosphorylation in TNF-a- stimulated neutrophils in vitro.
  • Neutrophils isolated from SCD patients (FIG. 13A) and mice (FIG. 13B) were pre-treated with 0.1 % DMSO (0), or 5, 50, or 500 nM ARQ 092, followed by stimulation with 20 ng/ml TNF-a.
  • Equal amounts (50 of cell lysate protein were immunoblotted. Representative blots were obtained from three independent experiments.
  • a further aspect includes from the one particular value and/or to the other particular value.
  • a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the disclosure.
  • the upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also encompassed within the disclosure, subject to any specifically excluded limit in the stated range.
  • the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure.
  • ranges excluding either or both of those included limits are also included in the disclosure, e.g. the phrase "x to y" includes the range from 'x' to 'y' as well as the range greater than 'x' and less than y.
  • the range can also be expressed as an upper limit, e.g. 'about x, y, z, or less' and should be interpreted to include the specific ranges of 'about x', 'about y', and 'about z' as well as the ranges of 'less than x', less than y', and 'less than z'.
  • the phrase 'about x, y, z, or greater' should be interpreted to include the specific ranges of 'about x', 'about y', and 'about z' as well as the ranges of 'greater than x', greater than y', and 'greater than z'.
  • the phrase "about 'x' to 'y'", where 'x' and y are numerical values, includes "about 'x' to about 'y'".
  • ratios, concentrations, amounts, and other numerical data can be expressed herein in a range format. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint. It is also understood that there are a number of values disclosed herein, and that each value is also herein disclosed as “about” that particular value in addition to the value itself. For example, if the value “10” is disclosed, then “about 10" is also disclosed. Ranges can be expressed herein as from “about” one particular value, and/or to "about” another particular value. Similarly, when values are expressed as approximations, by use of the antecedent "about,” it will be understood that the particular value forms a further aspect. For example, if the value "about 10" is disclosed, then “10” is also disclosed.
  • a numerical range of "about 0.1 % to 5%” should be interpreted to include not only the explicitly recited values of about 0.1 % to about 5%, but also include individual values (e.g., about 1 %, about 2%, about 3%, and about 4%) and the sub-ranges (e.g., about 0.5% to about 1.1 %; about 5% to about 2.4%; about 0.5% to about 3.2%, and about 0.5% to about 4.4%, and other possible sub-ranges) within the indicated range.
  • an amount, size, formulation, parameter or other quantity or characteristic is "about,” “approximate,” or “at or about” whether or not expressly stated to be such. It is understood that where "about,” “approximate,” or “at or about” is used before a quantitative value, the parameter also includes the specific quantitative value itself, unless specifically stated otherwise.
  • Embodiments of the present disclosure will employ, unless otherwise indicated, techniques of molecular biology, microbiology, organic chemistry, biochemistry, physiology, cell biology, pharmaceutical chemistry, and the like, which are within the skill of the art. Such techniques are explained fully in the literature.
  • active agent refers to a substance, compound, or molecule, which is biologically active or otherwise, induces a biological or physiological effect on a subject to which it is administered to.
  • active agent or “active ingredient” refers to a component or components of a composition to which the whole or part of the effect of the composition is attributed.
  • administering can refer to an administration that is oral, topical, intravenous, subcutaneous, transcutaneous, transdermal, intramuscular, intra-joint, parenteral, intra-arteriole, intradermal, intraventricular, intraosseous, intraocular, intracranial, intraperitoneal, intralesional, intranasal, intracardiac, intraarticular, intracavernous, intrathecal, intravireal, intracerebral, and intracerebroventricular, intratympanic, intracochlear, rectal, vaginal, by inhalation, by catheters, stents or via an implanted reservoir or other device that administers, either actively or passively (e.g.
  • a composition the perivascular space and adventitia.
  • a medical device such as a stent can contain a composition or formulation disposed on its surface, which can then dissolve or be otherwise distributed to the surrounding tissue and cells.
  • parenteral can include subcutaneous, intravenous, intramuscular, intra-articular, intra-synovial, intrasternal, intrathecal, intrahepatic, intralesional, and intracranial injections or infusion techniques.
  • agent can refer to any substance, compound, molecule, and the like, which can be biologically active or otherwise can induce a biological and/or physiological effect on a subject to which it is administered to.
  • An agent can be a primary active agent, or in other words, the component(s) of a composition to which the whole or part of the effect of the composition is attributed.
  • An agent can be a secondary agent, or in other words, the component(s) of a composition to which an additional part and/or other effect of the composition is attributed.
  • ARQ 092 means 3- ⁇ 3- ⁇ 4- ⁇ 1-Aminocydobutyi)phenyi)-5-phenyi-3H- imidazo ⁇ bjpyridin ⁇ -y pyridin ⁇ -amine.
  • the preparation and characterization of ARQ 092 is described in Lapierre, J- et al.. Discovery of 3-(3-(4-(1-Aminocyclobutyl)phenyl)-5-phenyl- 3H-imidazo[4,5-6]pyridin-2-yl)pyridin-2-amine (ARQ 092): An Orally Bioavailable, Selective, and Potent Allosteric AKT Inhibitor, J. Med. Chem., 2016, 59 (13), pp 6455-6469. "ARQ 092" also includes pharmaceutically acceptable salts thereof.
  • biocompatible refers to a material that along with any metabolites or degradation products thereof that are generally non-toxic to the recipient and do not cause any significant adverse effects to the recipient.
  • biocompatible materials are materials that do not elicit a significant inflammatory or immune response when administered to a patient.
  • biodegradable generally refers to a material that will degrade or erode under physiologic conditions to smaller units or chemical species that are capable of being metabolized, eliminated, or excreted by the subject.
  • the degradation time is a function of composition and morphology. Degradation times can be from hours to weeks.
  • control can refer to an alternative subject or sample used in an experiment for comparison purpose and included to minimize or distinguish the effect of variables other than an independent variable.
  • dose refers to physically discrete units suitable for use in a subject, each unit containing a predetermined quantity of ARQ 092 and/or hydroxyurea (HU) and/or a pharmaceutical formulation thereof calculated to produce the desired response or responses in association with its administration.
  • HU hydroxyurea
  • effective amount can refer to the amount of a compound provided herein that is sufficient to effect beneficial or desired biological, emotional, medical, or clinical response of a cell, tissue, system, animal, or human.
  • An effective amount can be administered in one or more administrations, applications, or dosages.
  • cam also include within its scope amounts effective to enhance or restore to substantially normal physiological function.
  • Effective amount can refer to the amount of ARQ 092 and/or Hydroxyurea (HU) described herein that can treat SCD or a symptom thereof.
  • Effective amount can refer to the amount of ARQ 092 and/or Hydroxyurea (HU) described herein that can eliminate or reduce a symptom of SCD.
  • Effective amount can be used to describe the amount ARQ 092 and/or Hydroxyurea (HU) that can decrease the activation state of neutrophils and/or platelets in a subject in need thereof. "Effective amount” can be used to describe the amount ARQ 092 and/or Hydroxyurea (HU) that can reduce the amount of platelet-neutrophil interaction in a subject in need thereof. “Effective amount” can be used to describe the amount ARQ 092 and/or Hydroxyurea (HU) that can reduce, block, or eliminate neutrophil-EC interactions in a subject in need thereof.
  • Effective amount can be used to describe the amount ARQ 092 and/or Hydroxyurea (HU) that can reduce, block, or eliminate neutrophil-platelet interactions in venules in a subject in need thereof.
  • Effective amount can be used to describe the amount ARQ 092 and/or Hydroxyurea (HU) that can reduce or impair neutrophil infiltration into the alveoli in a subject in need thereof.
  • Effective amount can be used to describe the amount ARQ 092 and/or Hydroxyurea (HU) that increase survival or extend the lifespan of a subject in need thereof.
  • Effectivee amount can be used to describe the amount of ARQ 092 that can reduce one or more side effects of HU when used as a co-therapy or in conjunction with HU.
  • the subject in need thereof can have, be predisposed to, and/or be suspected of having SCD.
  • hydroxyl urea also includes pharmaceutically acceptable salts thereof.
  • hydrophilic refers to substances that have strongly polar groups that are readily soluble in water.
  • hydrophobic refers to substances that lack an affinity for water; tending to repel and not absorb water as well as not dissolve in or mix with water.
  • mammal for the purposes of treatments, refers to any animal classified as a mammal, including human, domestic and farm animals, nonhuman primates, and zoo, sports, or pet animals, such as, but not limited to, dogs, horses, cats, and cows.
  • modulating refers to the treating, prevention, suppression, enhancement or induction of a function, condition or disorder.
  • SCD sickle cell disease
  • molecular weight can generally refer to the mass or average mass of a material. If a polymer or oligomer, the molecular weight can refer to the relative average chain length or relative chain mass of the bulk polymer. In practice, the molecular weight of polymers and oligomers can be estimated or characterized in various ways including gel permeation chromatography (GPC) or capillary viscometry. GPC molecular weights are reported as the weight-average molecular weight (M w ) as opposed to the number- average molecular weight (M n ). Capillary viscometry provides estimates of molecular weight as the inherent viscosity determined from a dilute polymer solution using a particular set of concentration, temperature, and solvent conditions.
  • organ refers to any living entity comprised of at least one cell.
  • a living organism can be as simple as, for example, a single isolated eukaryotic cell or cultured cell or cell line, or as complex as a mammal, including a human being, and animals (e.g., vertebrates, amphibians, fish, mammals, e.g., cats, dogs, horses, pigs, cows, sheep, rodents, rabbits, squirrels, bears, primates (e.g., chimpanzees, gorillas, and humans).
  • animals e.g., vertebrates, amphibians, fish, mammals, e.g., cats, dogs, horses, pigs, cows, sheep, rodents, rabbits, squirrels, bears, primates (e.g., chimpanzees, gorillas, and humans).
  • “Pharmaceutically acceptable salt” refers to any acid and/or base addition salt.
  • the acid or base addition salt can have counter-ions that are non-toxic to the subject to which they are administered in pharmaceutical doses of the salts.
  • pharmaceutical formulation refers to the combination of an active agent, compound, or ingredient with a pharmaceutically acceptable carrier or excipient, making the composition suitable for diagnostic, therapeutic, or preventive use in vitro, in vivo, or ex vivo.
  • the term "pharmaceutically acceptable vehicle” refers to a diluent, adjuvant, excipient or carrier with which a compound of the disclosure is administered.
  • the terms "effective amount” or “pharmaceutically effective amount” refer to a nontoxic but sufficient amount of the agent to provide the desired biological result. That result can be reduction and/or alleviation of the signs, symptoms, or causes of a disease, or any other desired alteration of a biological system.
  • An appropriate "effective” amount in any individual case can be determined by one of ordinary skill in the art using routine experimentation.
  • “Pharmaceutically acceptable carriers” for therapeutic use are well known in the pharmaceutical art, and are described, for example, in Remington's Pharmaceutical Sciences, 18th Edition (Easton, Pennsylvania: Mack Publishing Company, 1990).
  • sterile saline and phosphate-buffered saline at physiological pH can be used.
  • Preservatives, stabilizers, dyes and even flavoring agents can be provided in the pharmaceutical composition.
  • sodium benzoate, sorbic acid and esters of p- hydroxybenzoic acid can be added as preservatives. Id. at 1449.
  • antioxidants and suspending agents can be used. Id.
  • preventative can refer to hindering or stopping a disease or condition before it occurs, even if undiagnosed, or while the disease or condition is still in the sub-clinical phase.
  • subject refers to a warm blooded animal such as a mammal, preferably a human, or a human child, which is afflicted with, or has the potential and/or is suspected to be afflicted with one or more diseases and disorders described herein.
  • Subject can also refer to a cell, a population of cells, a tissue, an organ, or an organism, preferably to human and constituents thereof.
  • the terms “sufficient” and “effective,” can refer to an amount (e.g. mass, volume, dosage, concentration, and/or time period) needed to achieve one or more desired result(s).
  • a therapeutically effective amount refers to an amount needed to achieve one or more therapeutic effects.
  • “synergistic effect,” “synergism,” or “synergy” can refer to an effect arising between two or more molecules, compounds, substances, factors, or compositions that that is greater than or different from the sum of their individual effects.
  • Treating covers the treatment of a disease or disorder described herein (e.g. sickle cell disease (SCD)), in a subject, preferably a human, and includes:
  • SCD sickle cell disease
  • Treating can refer to both therapeutic treatment alone, prophylactic treatment alone, or both therapeutic and prophylactic treatment.
  • Those in need of treatment can include those already with the disorder and/or those in which the disorder is to be prevented.
  • the term “treating” can include inhibiting the disease, disorder or condition, e.g., impeding its progress; and relieving the disease, disorder, or condition, e.g., causing regression of the disease, disorder and/or condition.
  • Treating the disease, disorder, or condition can include reducing and/or ameliorating at least one symptom of the particular disease, disorder, or condition, even if the underlying pathophysiology is not affected, such as treating the pain of a subject by administration of an analgesic agent even though such agent does not treat the cause of the pain.
  • therapeutic can refer to treating, healing, and/or ameliorating a disease, disorder, condition, or side effect, or to decreasing in the rate of advancement of a disease, disorder, condition, or side effect.
  • a “therapeutically effective amount” can therefore refer to an amount of a compound that can yield a therapeutic effect.
  • Sickle cell disease is an inherited blood disorder caused by a homozygous Glu6Val mutation at the 6th position of ⁇ -globin (hemoglobin S (HbS)). HbS are polymerized upon deoxygenation, resulting in sickling and hemolysis of red blood cells, endothelial cell (EC) activation, and chronic inflammation.
  • HbS ⁇ °-thalassemia that is often clinically similar to sickle cell anemia.
  • recurrent vaso-occlusive episodes mediated by heterotypic cell-cell adhesion/aggregation cause pain crises and increase mortality due to organ damage and acute chest syndrome.
  • HU Hydroxyurea
  • NOx nitric oxide
  • compositions containing an effective amount of ARQ 092, HU, or ARQ 092 and HU that can be administered to a subject having, predisposed to developing, and/or suspected of having a SCD.
  • methods of treating a SCD in a subject in need thereof that can include administering to the subject an effective amount of ARQ 092 or a pharmaceutically acceptable salt thereof.
  • methods of treating a SCD in a subject in need thereof that can include administering an amount of ARQ 092 or a pharmaceutically acceptable salt thereof and an amount of HU or a pharmaceutically acceptable salt thereof to a subject in need thereof.
  • the subject in need thereof can have, be predisposed to developing, and/or suspected of having a SCD.
  • HU can be administered prior to administration of the ARQ 092.
  • Other compositions, compounds, methods, features, and advantages of the present disclosure will be or become apparent to one having ordinary skill in the art upon examination of the following drawings, detailed description, and examples. It is intended that all such additional compositions, compounds, methods, features, and advantages be included within this description, and be within the scope of the present disclosure.
  • Intravital microscopy supports that neutrophil-platelet interactions on activated ECs can cause microvascular occlusion under thromboinflammatory conditions, including SCD and ischemia/reperfusion injury.
  • neutrophil- platelet association is primarily mediated by the interaction of neutrophil P-selectin glycoprotein ligand-1 (PSGL-1) and ⁇ 2 integrin with platelet P-selectin and glycoprotein Iba (GPIba), respectively.
  • PSGL-1 neutrophil P-selectin glycoprotein ligand-1
  • GPIba glycoprotein Iba
  • AKT As a Ser/Thr protein kinase, AKT regulates numerous cellular processes, such as cell growth, survival, and metabolism. 14 Its activity is controlled by phosphorylation of the Thr308 and Ser473 residues by 3-phosphoinositide-dependent kinase 1 and mammalian target of rapamycin complex 2, respectively. Activated AKT then phosphorylates Ser/Thr residues in a variety of substrates. Despite 80% sequence homology of the three isoforms, each AKT isoform plays a partially overlapping but distinct role in platelet activation and aggregation.
  • AKT1 In neutrophils, which express AKT1 and AKT2, only AKT2 regulates cell migration, NADPH oxidase 2 (NOX2) activation, ⁇ 2 integrin function, and neutrophil-platelet interactions under inflammatory conditions.
  • NOX2 NADPH oxidase 2
  • ⁇ 2 integrin function As a major isoform in ECs, AKT1 modulates the activity of endothelial NO synthase (eNOS) and is involved in angiogenesis, acute inflammation, and atherosclerosis.
  • eNOS endothelial NO synthase
  • Human AKT isoforms share around 98% sequence homology with mouse proteins.
  • ARQ 092 has been reported as an orally-available, highly-selective AKT inhibitor. Recent studies show that ARQ 092 blocks the activity of AKT1 , AKT2, and AKT3 with an ICso value of 5.0, 4.5, and 16 nM, respectively, and that it shows excellent selectivity (>1 , 000-fold) over other kinases. 26 As an allosteric inhibitor, this compound blocks membrane translocation of inactive AKT and even dephosphorylates the membrane-associated active form, thereby perturbing AKT activity.
  • Described herein are pharmaceutical formulations that can include an amount, such as an effective amount to treat an SCD or symptom thereof in a subject in need thereof, of ARQ 092 and a pharmaceutically acceptable carrier.
  • the pharmaceutical formulation can also include an amount, such as an effective amount to treat an SCD or symptom thereof in a subject in need thereof of hydroxyurea (HU).
  • the pharmaceutical formulations described herein can be administered to a subject in need thereof.
  • the subject in need thereof can have SCD or a symptom thereof.
  • the ARQ 092 or ARQ 092 and HU can be formulated for parenteral delivery, such as injection or infusion, in the form of a solution or suspension.
  • the formulation can be administered via any route, such as, the blood stream or directly to the organ or tissue to be treated.
  • Parenteral formulations can be prepared as aqueous compositions using techniques is known in the art.
  • such compositions can be prepared as injectable formulations, for example, solutions or suspensions; solid forms suitable for using to prepare solutions or suspensions upon the addition of a reconstitution medium prior to injection; emulsions, such as water-in-oil (w/o) emulsions, oil-in-water (o/w) emulsions, and microemulsions thereof, liposomes, or emulsomes.
  • injectable formulations for example, solutions or suspensions
  • solid forms suitable for using to prepare solutions or suspensions upon the addition of a reconstitution medium prior to injection emulsions, such as water-in-oil (w/o) emulsions, oil-in-water (o/w) emulsions, and microemulsions thereof, liposomes, or emulsomes.
  • emulsions such as water-in-oil (w/o) emulsions
  • the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, one or more polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), oils, such as vegetable oils (e.g., peanut oil, corn oil, sesame oil, etc.), and combinations thereof.
  • polyols e.g., glycerol, propylene glycol, and liquid polyethylene glycol
  • oils such as vegetable oils (e.g., peanut oil, corn oil, sesame oil, etc.)
  • the proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and/or by the use of surfactants.
  • isotonic agents for example, sugars or sodium chloride.
  • Solutions and dispersions of the ARQ 092 or ARQ 092 and HU described herein can be prepared in water or another solvent or dispersing medium suitably mixed with one or more pharmaceutically acceptable excipients including, but not limited to, surfactants, dispersants, emulsifiers, pH modifying agents, and combination thereof.
  • Suitable surfactants can be anionic, cationic, amphoteric or nonionic surface active agents.
  • Suitable anionic surfactants include, but are not limited to, those containing carboxylate, sulfonate and sulfate ions.
  • Suitable anionic surfactants include sodium, potassium, ammonium of long chain alkyl sulfonates and alkyl aryl sulfonates such as sodium dodecylbenzene sulfonate; dialkyl sodium sulfosuccinates, such as sodium dodecylbenzene sulfonate; dialkyl sodium sulfosuccinates, such as sodium bis-(2-ethylthioxyl)-sulfosuccinate; and alkyl sulfates such as sodium lauryl sulfate.
  • Suitable cationic surfactants include, but are not limited to, quaternary ammonium compounds such as benzalkonium chloride, benzethonium chloride, cetrimonium bromide, stearyl dimethylbenzyl ammonium chloride, polyoxyethylene and coconut amine.
  • Suitable nonionic surfactants include ethylene glycol monostearate, propylene glycol myristate, glyceryl monostearate, glyceryl stearate, polyglyceryl-4-oleate, sorbitan acylate, sucrose acylate, PEG- 150 laurate, PEG-400 monolaurate, polyoxyethylene monolaurate, polysorbates, polyoxyethylene octylphenylether, PEG-1000 cetyl ether, polyoxyethylene tridecyl ether, polypropylene glycol butyl ether, Poloxamer® 401 , stearoyl monoisopropanolamide, and polyoxyethylene hydrogenated tallow amide.
  • amphoteric surfactants include sodium N-dodecyl ⁇ -alanine, sodium N- lauryl ⁇ -iminodipropionate, myristoamphoacetate, lauryl betaine and lauryl sulfobetaine.
  • the pharmaceutical formulation can contain a preservative to prevent the growth of microorganisms. Suitable preservatives include, but are not limited to, parabens, chlorobutanol, phenol, sorbic acid, and thimerosal.
  • the pharmaceutical formulation can also contain an antioxidant to prevent degradation of the self-assembling cyclopeptide-dye compound(s).
  • the pharmaceutical formulation can be buffered to a pH of 3-8 for parenteral administration upon reconstitution.
  • Suitable buffers include, but are not limited to, phosphate buffers, acetate buffers, and citrate buffers.
  • Water-soluble polymers can be used in the formulations for parenteral administration. Suitable water-soluble polymers include, but are not limited to, polyvinylpyrrolidone, dextran, carboxymethylcellulose, and polyethylene glycol.
  • Sterile injectable solutions can be prepared by incorporating the neruosteroid(s) and/or analogue(s) thereof in the desired amount in the appropriate solvent or dispersion medium with one or more of the excipients listed above, as required, followed by filtered sterilization.
  • Dispersions can be prepared by incorporating the various sterilized neruosteroid(s) and/or analogue(s) thereof into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those listed above.
  • Sterile powders for the preparation of sterile injectable solutions can be prepared by vacuum-drying and freeze-drying techniques, which yields a powder of the ARQ 092 or ARQ 092 and HU described herein with or without any additional desired ingredient from a previously sterile- filtered solution thereof.
  • the powders can be prepared in such a manner that the particles are porous in nature, which can increase dissolution of the particles. Methods for making porous particles are well known in the art.
  • Pharmaceutical formulations for parenteral administration can be in the form of a sterile aqueous solution or suspension of the ARQ 092 or ARQ 092 and HU described herein.
  • Acceptable solvents include, for example, water, Ringer's solution, phosphate buffered saline (PBS), and isotonic sodium chloride solution.
  • the formulation can also be a sterile solution, suspension, or emulsion in a nontoxic, parenterally acceptable diluent or solvent such as 1 ,3- butanediol.
  • the formulation can be distributed or packaged in a liquid form.
  • formulations for parenteral administration can be packed as a solid, obtained, for example by lyophilization of a suitable liquid formulation.
  • the solid can be reconstituted with an appropriate carrier or diluent prior to administration.
  • Solutions, suspensions, or emulsions for parenteral administration can be buffered with an effective amount of buffer necessary to maintain a pH suitable for ocular administration.
  • Suitable buffers include, but are not limited to, acetate, borate, carbonate, citrate, and phosphate buffers.
  • Solutions, suspensions, or emulsions for parenteral administration can also contain one or more tonicity agents to adjust the isotonic range of the formulation.
  • Suitable tonicity agents include, but are not limited to, glycerin, mannitol, sorbitol, sodium chloride, and other electrolytes.
  • Solutions, suspensions, or emulsions for parenteral administration can also contain one or more preservatives to prevent bacterial contamination of the ophthalmic preparations.
  • Suitable preservatives include, but are not limited to, polyhexamethylenebiguanidine (PHMB), benzalkonium chloride (BAK), stabilized oxychloro complexes (otherwise known as Purite®), phenylmercuric acetate, chlorobutanol, sorbic acid, chlorhexidine, benzyl alcohol, parabens, thimerosal, and mixtures thereof.
  • Solutions, suspensions, or emulsions, including nanoformulations for parenteral administration can also contain one or more excipients, such as dispersing agents, wetting agents, and suspending agents.
  • the ARQ 092 or ARQ 092 and HU described herein can be formulated for topical administration.
  • Suitable dosage forms for topical administration include creams, ointments, salves, sprays, gels, lotions, emulsions, liquids, and transdermal patches.
  • the formulation can be formulated for transmucosal, transepithelial, transendothelial, or transdermal administration.
  • the topical formulations can contain one or more chemical penetration enhancers, membrane permeability agents, membrane transport agents, emollients, surfactants, stabilizers, and combination thereof.
  • the ARQ 092 or ARQ 092 and HU described herein can be administered as a liquid formulation, such as a solution or suspension, a semi-solid formulation, such as a lotion or ointment, or a solid formulation.
  • a liquid formulation such as a solution or suspension
  • a semi-solid formulation such as a lotion or ointment
  • a solid formulation such as a liquid formulation, such as a solution or suspension
  • the ARQ 092 or ARQ 092 and HU described herein can be formulated as liquids, including solutions and suspensions, such as eye drops or as a semi-solid formulation, such as ointment or lotion for topical application to the skin, to the mucosa, such as the eye, to the vagina, or to the rectum.
  • the pharmaceutical formulation can contain one or more excipients, such as emollients, surfactants, emulsifiers, penetration enhancers, and the like.
  • excipients such as emollients, surfactants, emulsifiers, penetration enhancers, and the like.
  • Suitable emollients include, without limitation, almond oil, castor oil, ceratonia extract, cetostearoyl alcohol, cetyl alcohol, cetyl esters wax, cholesterol, cottonseed oil, cyclomethicone, ethylene glycol palmitostearate, glycerin, glycerin monostearate, glyceryl monooleate, isopropyl myristate, isopropyl palmitate, lanolin, lecithin, light mineral oil, medium-chain triglycerides, mineral oil and lanolin alcohols, petrolatum, petrolatum and lanolin alcohols, soybean oil, starch, stearyl alcohol, sunflower oil, xylitol and combinations thereof.
  • the emollients can be ethylhexylstearate and ethylhexyl palmitate.
  • Suitable surfactants include, but are not limited to, emulsifying wax, glyceryl monooleate, polyoxyethylene alkyl ethers, polyoxyethylene castor oil derivatives, polysorbate, sorbitan esters, benzyl alcohol, benzyl benzoate, cyclodextrins, glycerin monostearate, poloxamer, povidone and combinations thereof.
  • the surfactant can be stearyl alcohol.
  • Suitable emulsifiers include, but are not limited to, acacia, metallic soaps, certain animal and vegetable oils, and various polar compounds, anionic emulsifying wax, calcium stearate, carbomers, cetostearyl alcohol, cetyl alcohol, cholesterol, diethanolamine, ethylene glycol palmitostearate, glycerin monostearate, glyceryl monooleate, hydroxpropyl cellulose, hypromellose, lanolin, hydrous, lanolin alcohols, lecithin, medium-chain triglycerides, methylcellulose, mineral oil and lanolin alcohols, monobasic sodium phosphate, monoethanolamine, nonionic emulsifying wax, oleic acid, poloxamer, poloxamers, polyoxyethylene alkyl ethers, polyoxyethylene castor oil derivatives, polyoxyethylene sorbitan fatty acid esters, polyoxyethylene stearates, propylene glycol alginate, self-emulsifying
  • Suitable classes of penetration enhancers include, but are not limited to, fatty alcohols, fatty acid esters, fatty acids, fatty alcohol ethers, amino acids, phospholipids, lecithins, cholate salts, enzymes, amines and amides, complexing agents (liposomes, cyclodextrins, modified celluloses, and diimides), macrocyclics, such as macrocylic lactones, ketones, and anhydrides and cyclic ureas, surfactants, N-methyl pyrrolidones and derivatives thereof, DMSO and related compounds, ionic compounds, azone and related compounds, and solvents, such as alcohols, ketones, amides, polyols (e.g., glycols).
  • Suitable emulsions include, but are not limited to, oil-in-water and water-in-oil emulsions. Either or both phases of the emulsions can include a surfactant, an emulsifying agent, and/or a liquid non-volatile non-aqueous material.
  • the surfactant can be a non-ionic surfactant.
  • the emulsifying agent can be an emulsifying wax.
  • the liquid non-volatile non-aqueous material is a glycol. In some aspects, the glycol is propylene glycol.
  • the oil phase can contain other suitable oily pharmaceutically acceptable excipients. Suitable oily pharmaceutically acceptable excipients include, but are not limited to, hydroxylated castor oil or sesame oil can be used in the oil phase as surfactants or emulsifiers.
  • Lotions containing the ARQ 092 or ARQ 092 and HU described herein are also described herein.
  • the lotion can be in the form of an emulsion having a viscosity of between 100 and 1000 centistokes.
  • the fluidity of lotions can permit rapid and uniform application over a wide surface area.
  • Lotions can be formulated to dry on the skin leaving a thin coat of their medicinal components on the skin's surface.
  • Creams containing the ARQ 092 or ARQ 092 and HU described herein are also described herein.
  • the cream can contain emulsifying agents and/or other stabilizing agents.
  • the cream can be in the form of a cream having a viscosity of greater than 1000 centistokes, typically in the range of 20,000-50,000 centistokes. Creams, as compared to ointments, can be easier to spread and easier to remove.
  • Creams can be thicker than lotions, can have various uses, and can have more varied oils/butters, depending upon the desired effect upon the skin.
  • the water-base percentage can be about 60% to about 75% and the oil-base can be about 20% to about 30% of the total, with the other percentages being the emulsifier agent, preservatives and additives for a total of 100%.
  • Ointments containing the ARQ 092 or ARQ 092 and HU described herein and a suitable ointment base are also provided.
  • Suitable ointment bases can include hydrocarbon bases (e.g., petrolatum, white petrolatum, yellow ointment, and mineral oil); absorption bases (hydrophilic petrolatum, anhydrous lanolin, lanolin, and cold cream); water-removable bases (e.g., hydrophilic ointment), and water-soluble bases (e.g., polyethylene glycol ointments).
  • Pastes typically differ from ointments in that they contain a larger percentage of solids. Pastes are typically more absorptive and less greasy that ointments prepared with the same components.
  • gels that can contain the ARQ 092 or ARQ 092 and HU described herein, a gelling agent, and a liquid vehicle.
  • Suitable gelling agents include, but are not limited to, modified celluloses, such as hydroxypropyl cellulose and hydroxyethyl cellulose; carbopol homopolymers and copolymers; thermoreversible gels and combinations thereof.
  • Suitable solvents in the liquid vehicle can include, but are not limited to, diglycol monoethyl ether; alklene glycols, such as propylene glycol; dimethyl isosorbide; alcohols, such as isopropyl alcohol and ethanol. The solvents can be selected for their ability to dissolve the drug.
  • additives which can improve the skin feel and/or emolliency of the formulation, can also be incorporated.
  • Such additives include, but are not limited, isopropyl myristate, ethyl acetate, C12-C15 alkyl benzoates, mineral oil, squalane, cyclomethicone, capric/caprylic triglycerides, and combinations thereof.
  • foams that can include the ARQ 092 or ARQ 092 and HU described herein.
  • Foams can be an emulsion in combination with a gaseous propellant.
  • the gaseous propellant can include hydrofluoroalkanes (HFAs).
  • HFAs hydrofluoroalkanes
  • Suitable propellants include HFAs such as 1 , 1 , 1 ,2-tetrafluoroethane (HFA 134a) and 1 , 1 ,1 ,2,3,3,3-heptafluoropropane (HFA 227), but mixtures and admixtures of these and other HFAs that are currently approved or can become approved for medical use are suitable.
  • the propellants can be devoid of hydrocarbon propellant gases, which can produce flammable or explosive vapors during spraying.
  • the foams can contain no volatile alcohols, which can produce flammable or explosive vapors during use.
  • Buffers can be used to control pH of a composition.
  • the buffers can buffer the composition from a pH of about 4 to a pH of about 7.5, from a pH of about 4 to a pH of about 7, or from a pH of about 5 to a pH of about 7.
  • the buffer can be triethanolamine.
  • Preservatives can be included to prevent the growth of fungi and microorganisms.
  • Suitable preservatives can include, but are not limited to, benzoic acid, butylparaben, ethyl paraben, methyl paraben, propylparaben, sodium benzoate, sodium propionate, benzalkonium chloride, benzethonium chloride, benzyl alcohol, cetylpyridinium chloride, chlorobutanol, phenol, phenylethyl alcohol, and thimerosal.
  • the pharmaceutical formulations can be provided via continuous delivery of one or more formulations to a subject in need thereof.
  • repeated application can be done or a patch can be used to provide continuous administration of the noscapine analogs over an extended period of time.
  • tARQ 092 or ARQ 092 and HU described herein can be prepared in enteral formulations, such as for oral administration.
  • Suitable oral dosage forms include tablets, capsules, solutions, suspensions, syrups, and lozenges. Tablets can be made using compression or molding techniques well known in the art.
  • Gelatin or non-gelatin capsules can prepared as hard or soft capsule shells, which can encapsulate liquid, solid, and semi-solid fill materials, using techniques well known in the art.
  • Formulations containing the ARQ 092 or ARQ 092 and HU described herein can be prepared using pharmaceutically acceptable carriers.
  • carrier includes, but is not limited to, diluents, preservatives, binders, lubricants, disintegrators, swelling agents, fillers, stabilizers, and combinations thereof.
  • Polymers used in the dosage form include, but are not limited to, suitable hydrophobic or hydrophilic polymers and suitable pH dependent or independent polymers.
  • Suitable hydrophobic and hydrophilic polymers include, but are not limited to, hydroxypropyl methylcellulose, hydroxypropyl cellulose, hydroxyethyl cellulose, carboxy methylcellulose, polyethylene glycol, ethylcellulose, microcrystalline cellulose, polyvinyl pyrrolidone, polyvinyl alcohol, polyvinyl acetate, and ion exchange resins.
  • Carrier can also include all components of the coating composition which can include plasticizers, pigments, colorants, stabilizing agents, and glidants.
  • Formulations containing the ARQ 092 or ARQ 092 and HU described herein can be prepared using one or more pharmaceutically acceptable excipients, including diluents, preservatives, binders, lubricants, disintegrators, swelling agents, fillers, stabilizers, and combinations thereof.
  • Delayed release dosage formulations containing the ARQ 092 or ARQ 092 and HU described herein can be prepared as described in standard references such as "Pharmaceutical dosage form tablets”, eds. Liberman et. al. (New York, Marcel Dekker, Inc., 1989), "Remington - The science and practice of pharmacy", 20th ed., Lippincott Williams & Wilkins, Baltimore, MD, 2000, and "Pharmaceutical dosage forms and drug delivery systems", 6th Edition, Ansel et al., (Media, PA: Williams and Wlkins, 1995). These references provide information on excipients, materials, equipment and process for preparing tablets and capsules and delayed release dosage forms of tablets, capsules, and granules. These references provide information on carriers, materials, equipment and process for preparing tablets and capsules and delayed release dosage forms of tablets, capsules, and granules.
  • the formulations containing the ARQ 092 or ARQ 092 and HU described herein can be coated with a suitable coating material, for example, to delay release once the particles have passed through the acidic environment of the stomach.
  • suitable coating materials include, but are not limited to, cellulose polymers such as cellulose acetate phthalate, hydroxypropyl cellulose, hydroxypropyl methylcellulose, hydroxypropyl methylcellulose phthalate and hydroxy propyl methylcellulose acetate succinate; polyvinyl acetate phthalate, acrylic acid polymers and copolymers, and methacrylic resins that are commercially available under the trade name EUDRAGIT® (Roth Pharma, Westerstadt, Germany), zein, shellac, and polysaccharides.
  • Coatings can be formed with a different ratio of water soluble polymer, water insoluble polymers and/or pH dependent polymers, with or without water insoluble/water soluble non polymeric excipient, to produce the desired release profile.
  • the coating can be performed on a dosage form (matrix or simple) which includes, but is not limited to, tablets (compressed with or without coated beads), capsules (with or without coated beads), beads, particle compositions, "ingredient as is” formulated as, but not limited to, suspension form or as a sprinkle dosage form.
  • the coating material can contain conventional carriers such as plasticizers, pigments, colorants, glidants, stabilization agents, pore formers and surfactants.
  • Optional pharmaceutically acceptable excipients include, but are not limited to, diluents, binders, lubricants, disintegrants, colorants, stabilizers, and surfactants.
  • Diluents also referred to as "fillers,” can be used to increase the bulk of a solid dosage form so that a practical size is provided for compression of tablets or formation of beads and granules.
  • Suitable diluents include, but are not limited to, dicalcium phosphate dihydrate, calcium sulfate, lactose, sucrose, mannitol, sorbitol, cellulose, microcrystalline cellulose, kaolin, sodium chloride, dry starch, hydrolyzed starches, pregelatinized starch, silicone dioxide, titanium oxide, magnesium aluminum silicate and powdered sugar.
  • the usual diluents include inert powdered substances such as starches, powdered cellulose, especially crystalline and microcrystalline cellulose, sugars such as fructose, mannitol and sucrose, grain flours and similar edible powders.
  • Typical diluents include, for example, various types of starch, lactose, mannitol, kaolin, calcium phosphate or sulfate, inorganic salts such as sodium chloride and powdered sugar. Powdered cellulose derivatives are also useful.
  • Binders can impart cohesive qualities to a solid dosage formulation, and thus can ensure that a tablet or bead or granule remains intact after the formation of the dosage forms.
  • Suitable binder materials include, but are not limited to, starch, pregelatinized starch, gelatin, sugars (including sucrose, glucose, dextrose, lactose and sorbitol), polyethylene glycol, waxes, natural and synthetic gums such as acacia, tragacanth, sodium alginate, cellulose, including hydroxypropylmethylcellulose, hydroxypropylcellulose, ethylcellulose, and veegum, and synthetic polymers such as acrylic acid and methacrylic acid copolymers, methacrylic acid copolymers, methyl methacrylate copolymers, aminoalkyl methacrylate copolymers, polyacrylic acid/polymethacrylic acid and polyvinylpyrrolidone.
  • Typical tablet binders include substances such as starch, gelatin and sugars such as lactose, fructose, and glucose. Natural and synthetic gums, including acacia, alginates, methylcellulose, and polyvinylpyrrolidone can also be used. Polyethylene glycol, hydrophilic polymers, ethylcellulose and waxes can also serve as binders.
  • Lubricants can be included to facilitate tablet manufacture. Suitable lubricants include, but are not limited to, magnesium stearate, calcium stearate, stearic acid, glycerol behenate, polyethylene glycol, talc, and mineral oil. A lubricant can be included in a tablet formulation to prevent the tablet and punches from sticking in the die. The lubricant can be chosen from such slippery solids as talc, magnesium and calcium stearate, stearic acid and hydrogenated vegetable oils.
  • Disintegrants can be used to facilitate dosage form disintegration or "breakup" after administration, and generally include, but are not limited to, starch, sodium starch glycolate, sodium carboxymethyl starch, sodium carboxymethylcellulose, hydroxypropyl cellulose, pregelatinized starch, clays, cellulose, alginine, gums or cross linked polymers, such as cross- linked PVP (Polyplasdone® XL from GAF Chemical Corp).
  • PVP Polyplasdone® XL from GAF Chemical Corp.
  • Stabilizers can be used to inhibit or retard drug decomposition reactions which include, by way of example, oxidative reactions.
  • Suitable stabilizers include, but are not limited to, antioxidants, butylated hydroxytoluene (BHT); ascorbic acid, its salts and esters; Vitamin E, tocopherol and its salts; sulfites such as sodium metabisulphite; cysteine and its derivatives; citric acid; propyl gallate, and butylated hydroxyanisole (BHA).
  • the pharmaceutical formulation containaing ARQ 092 or ARQ 092 and HU described herein described herein can be presented as a combination kit.
  • the terms "combination kit” or “kit of parts” refers to the compounds, or pharmaceutical formulations and additional components that are used to package, sell, market, deliver, and/or administer the combination of elements or a single element, such as the active ingredient, contained therein.
  • additional components include, but are not limited to, packaging, syringes, blister packages, bottles, and the like.
  • the combination kit can contain the active agents in a single pharmaceutical formulation (e.g. a tablet) or in separate pharmaceutical formulations.
  • the combination kit can contain each active agent in separate pharmaceutical formulations.
  • the separate pharmaceutical formulations can be contained in a single package or in separate packages within the kit.
  • the kit can also include instructions and/oror indications for use printed on or otherwise contained in a tangible medium of expression. Instructions and/or indications for use can be incorporated in labels, boxes, containers, syrniges, delivery devices, insert sheets of paper, flash drives, CD-ROM, an internet website and the like.
  • the instructions can provide information regarding the content of the compound or pharmaceutical formulations contained therein, safety information regarding the content of the compound(s) or pharmaceutical formulation(s) contained therein, information regarding the dosages, indications for use, and/or recommended treatment regimen(s) for the compound(s) and/or pharmaceutical formulations contained therein.
  • the instructions provide directions for administering the compounds, compositions, pharmaceutical formulations, or salts thereof to a subject having, suspected of having, or predisposed to or at risk of developing SCD.
  • the instructions can provide directions for administering the pharmaceutical formulations containing an amount of ARQ 092 or ARQ 092 and HU, such as an amount effective to treat a SCD, described herein to a subject having, suspected of having, or predisposed to developing a SCD.
  • a pharmaceutical formulation containing an amount, such as an effective amount, of ARQ 092 or an amount of ARQ 092 and an amount of HU, such as an effective amount of each of ARQ 092 and HU as described herein, can be administered to a subject in need thereof.
  • both ARQ 092 and HU are administered, both the ARQ 092 and HU are contained in the same pharmaceutical formulation.
  • the ARQ 092 and HU are contained in separate pharmaceutical formulations that can be administered at about the same time or at different times during a treatment mitine.
  • the subject in need thereof can have, be suspected of having, and/or be predisposed to and/or at risk of developing a SCD.
  • the method can include administering an amount ARQ 092 or an amount of ARQ 092 and amount of HU.
  • the administration of both ARQ 092 and HU occurs at the same (e.g. when both are contained in the same pharmaceutical formulation) or substantially the same time (e.g. when the compounds are contained in separate pharmaceutical formulations but administered about at the same time). This can also be referred to as simultaneous administration.
  • both ARQ 092 and HU are administered, they are administered at different times.
  • the HU is administered prior to administration of the ARQ 092.
  • the amount of the ARQ 092 or HU can be an effective amount.
  • the ARQ 092 and HU can be contained in a pharmaceutical formulation.
  • the subject in need thereof can have, be suspected of having, be predisposed to, and/or at risk for developing a SCD.
  • the effective amount can reduce or eliminate a symptom of a SCD.
  • the pharmaceutical formulation(s) containing the ARQ 092, HU, or ARQ 092 and HU can be co-administered or be a co-therapy as part of a treatment regimen with another active agent or ingredient that can be included in the formulation or provided in a dosage form separate from the ARQ 092, HU, or ARQ 092 and HU and/or pharmaceutical formulation(s) thereof.
  • the amount of the ARQ 092 or HU administered can each range from about 0.1 ⁇ g/kg to up to about 1000 mg/kg or more, depending on the factors mentioned elsewhere herein. In certain aspects, the amount can range from 0.1 ⁇ g/kg up to about 500 mg/kg, or 1 ⁇ g/kg up to about 500 mg/kg, 5 ⁇ g/kg up to about 500 mg/kg, 0.1 ⁇ g/kg up to about 100 mg/kg, or 1 ⁇ g/kg up to about 100 mg/kg, 5 ⁇ g/kg up to about 100 mg/kg.
  • the effective amount of ARQ 092 can range from about 0.01 , 1 , 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 1 10, 120, 130, 140, 150, 160, 170, 180, 190, 20, 210, 220, 230, or 240 mg/kg to about 250 mg/kg body weight. In some aspects, the effective amount of ARQ 092 can range from about 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, or 140 mg/kg to about 150 mg/kg body weight.
  • the effective amount of HU can range from about 0.01 , 1 , 5, 10, 15, 20, 25, 30, 35, 40, or 45 mg/kg to about 50 mg/kg body weight. In some aspects, the effective amount of HU can range from about 10 15, 20, 25, 30, 35, 40, or 45 mg/kg to about 50 mg/kg body weight.
  • Administration of the ARQ 092, HU, or ARQ 092 and HU and/or pharmaceutical formulation(s) thereof can be systemic or localized.
  • the ARQ 092, HU, or ARQ 092 and HU and/or pharmaceutical formulation(s) thereof can be administered to the subject in need thereof one or more times per hour or day.
  • the ARQ 092, HU, or ARQ 092 and HU and/or pharmaceutical formulation(s) thereof can be administered once daily.
  • the ARQ 092, HU, or ARQ 092 and HU and/or pharmaceutical formulation(s) thereof can be administered can be administered 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, or more times daily.
  • an effective amount of the ARQ 092, HU, or ARQ 092 and HU and/or pharmaceutical formulation(s) thereof can be administered to the subject in need thereof.
  • the ARQ 092, HU, or ARQ 092 and HU and/or pharmaceutical formulation(s) thereof can be administered one or more times per week.
  • the ARQ 092, HU, or ARQ 092 and HU and/or pharmaceutical formulation(s) thereof can be administered 1 , 2, 3, 4, 5, 6 or 7 days per week.
  • the ARQ 092, HU, or ARQ 092 and HU and/or pharmaceutical formulation(s) thereof can be administered 1 , 2, 3, 4, 5, 6, 7, 8,, 9, 10, 1 1 , 12, or more times per month.
  • the ARQ 092, HU, or ARQ 092 and HU and/or pharmaceutical formulation(s) thereof can be administered 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, or more time per year.
  • the ARQ 092, HU, or ARQ 092 and HU and/or pharmaceutical formulation(s) thereof can be administered in a dosage form.
  • the amount or effective amount of the ARQ 092, HU, or ARQ 092 and HU and/or pharmaceutical formulation(s) thereof can be divided into multiple dosage forms.
  • the amount or effective amount can be split into two dosage forms and the one dosage forms can be administered, for example, in the morning, and the second dosage form can be administered in the evening.
  • the amount can be given over two or more doses, in one day, the subject can receives the desired amount or effective amount when the total amount administered across all the doses is considered.
  • the dosages can range from about 0.1 ⁇ g/kg to up to about 1000 mg/kg or more, depending on the factors mentioned above.
  • the dosage can range from 0.1 ⁇ g/kg up to about 500 mg/kg, or 1 ⁇ g/kg up to about 500 mg/kg, 5 ⁇ g/kg up to about 500 mg/kg, 0.1 ⁇ g/kg up to about 100 mg/kg, or 1 ⁇ g/kg up to about 100 mg/kg, 5 ⁇ g/kg up to about 100 mg/kg.
  • the dosage can contain an effective amount of the ARQ 092, HU, or ARQ 092 and HU.
  • the effective amount of ARQ 092 can range from about 0.01 , 1 , 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 1 10, 120, 130, 140, 150, 160, 170, 180, 190, 20, 210, 220, 230, or 240 mg/kg to about 250 mg/kg body weight. In some aspects, the effective amount of ARQ 092 can range from about 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 1 10, 120, 130, or 140 mg/kg to about 150 mg/kg body weight.
  • the effective amount of HU can range from about 0.01 , 1 , 5, 10, 15, 20, 25, 30, 35, 40, or 45 mg/kg to about 50 mg/kg body weight. In some aspects, the effective amount of HU can range from about 10 15, 20, 25, 30, 35, 40, or 45 mg/kg to about 50 mg/kg body weight.
  • Compound ARQ 092 for use in the treatmet of sickle cell disease.
  • Sickle cell disease is an inherited blood disorder caused by a homozygous Glu6Val mutation at the 6th position of ⁇ -globin (hemoglobin S (HbS)). HbS are polymerized upon deoxygenation, resulting in sickling and hemolysis of red blood cells, endothelial cell (EC) activation, and chronic inflammation. 1
  • heterozygous forms of SCD 2 such as HbS ⁇ °-thalassemia that is often clinically similar to sickle cell anemia.
  • recurrent vaso-occlusive episodes mediated by heterotypic cell-cell adhesion/aggregation cause pain crises and increase mortality due to organ damage and acute chest syndrome.
  • Intravital microscopy supports that neutrophil-platelet interactions on activated ECs can cause microvascular occlusion under thromboinflammatory conditions, including SCD and ischemia/reperfusion injury.
  • neutrophil- platelet association is primarily mediated by the interaction of neutrophil P-selectin glycoprotein ligand-1 (PSGL-1) and ⁇ 2 integrin with platelet P-selectin and glycoprotein Iba (GPIba), respectively.
  • PSGL-1 neutrophil P-selectin glycoprotein ligand-1
  • GPIba glycoprotein Iba
  • AKT2 inhibition may be a supplemental therapy for SCD patients with vaso-occlusive crises, no AKT2 specific inhibitor is currently available in the clinic.
  • Ser/Thr protein kinase AKT regulates numerous cellular processes, such as cell growth, survival, and metabolism. 14 Its activity is controlled by phosphorylation of the Thr308 and Ser473 residues by 3-phosphoinositide-dependent kinase 1 and mammalian target of rapamycin complex 2, respectively. 15 Activated AKT then phosphorylates Ser/Thr residues in a variety of substrates.
  • each AKT isoform plays a partially overlapping but distinct role in platelet activation and aggregation. 17" 9 In neutrophils, which express AKT1 and AKT2, only AKT2 regulates cell migration, NADPH oxidase 2 (NOX2) activation, ⁇ 2 integrin function, and neutrophil-platelet interactions under inflammatory conditions. 12 20 As a major isoform in ECs, AKT1 modulates the activity of endothelial NO synthase (eNOS) and is involved in angiogenesis, acute inflammation, and atherosclerosis. 21-23 Human AKT isoforms share around 98% sequence homology with mouse proteins. These studies suggest the importance of each AKT isoform in the pathophysiology of vascular diseases.
  • endothelial NO synthase eNOS
  • ARQ 092 blocks the activity of AKT1 , AKT2, and AKT3 with an ICso value of 5.0, 4.5, and 16 nM, respectively, and that it shows excellent selectivity (>1 , 000-fold) over other kinases.
  • this compound blocks membrane translocation of inactive AKT and even dephosphorylates the membrane-associated active form, thereby perturbing AKT activity.
  • mice C57BL/6, 6-week old, male and female
  • hemizygous Tg(Hu-minil_CRa1 G y A y6 s ) Hba- Hbb +
  • Berkeley sickle Tg(Hu-minil_CRa1 G y A y6 s ) Hba+ Hbb- mice were obtained from The Jackson Laboratory (Bar Harbor, ME).
  • SCD mice 21-24 weeks old were generated by transplantation of bone marrow cells isolated from Berkeley mice into lethally irradiated WT mice as described previously.
  • PCR and electrophoresis analyses showed that all chimeric mice expressed the transgene (human HbS) (FIGS.
  • SCD mice chimeric Berkeley mice were hereafter referred to as SCD mice. Both male and female SCD mice (20-24 weeks old) were used in this study. The University of Illinois Institutional Animal Care and Use Committee approved all animal care and experimental procedures. SCD patients. Sixteen homozygous (HbSS) and six HbS ⁇ °-thalassemia patients (20- 52 years, 9 men and 13 women) who did not take aspirin or ibuprofen within 5 days were included in our studies. None of the patients were treated with HU prior to blood donation. No significant differences were observed in the levels of surface markers of resting and stimulated platelets and neutrophils in patients with HbSS or HbS ⁇ °-thalassemia.
  • ARQ 092 preparation The synthesis process and molecular properties of ARQ 092 have been reported. 30
  • Intravital microscopy Male SCD mice were fasted overnight and treated with saline or 250 mg/kg of HU (50 mg/ml) by oral gavage and subsequently with ip injection of TNF-a (500 ng), 3 hours prior to imaging. Phosphoric acid (0.01 M) or ARQ 092 (100 mg/10 ml/kg) was administered orally 30 minutes before imaging. Platelets and neutrophils were monitored via infusion of DyLight 488-conjugated anti-CD42c (0.1 ⁇ g/g body weight (BW)) and Alexa Fluor 647-conjugated anti-Ly-6G antibodies (0.05 ⁇ g/g BW), respectively.
  • DyLight 488-conjugated anti-CD42c 0.1 ⁇ g/g body weight (BW)
  • Alexa Fluor 647-conjugated anti-Ly-6G antibodies 0.05 ⁇ g/g BW
  • Fluorescence and bright- field images were recorded using an Olympus BX61W microscope with a 60 ⁇ 1.0 NA water immersion objective and a Hamamatsu C9300 high-speed camera through an intensifier (Video Scope International), and data were analyzed using Slidebook v6.0 (Intelligent Imaging Innovations). Real-time images were captured in the inflamed cremaster venules with a diameter of 25-40 ⁇ . The rolling influx of neutrophils (rolling cells/minute) and number of adherent neutrophils were determined over a 5-minute period (number/field/5 minutes). Five to six different venules were monitored in each mouse.
  • ARQ 092 can inhibit activation of neutrophils and platelets isolated from SCD patients. It was reported that the basal phosphorylation levels of all AKT isoforms are significantly elevated in neutrophils and platelets isolated from SCD patients under stable conditions, compared with those from healthy donors. 12 It was found that AKT in human neutrophils and platelets is maximally phosphorylated 2 minutes after stimulation with fMLP or thrombin, respectively (FIGS. 9A-9B). Similar results were obtained with mouse neutrophils and platelets (FIGS. 9C-9D).
  • ARQ 092 Assay for AKT phosphorylation, neutrophils isolated from SCD patients were pretreated with ARQ 092 and further incubated with or without fMLP for 2 minutes, followed by immunoblotting. It was observed that ARQ 092 at 50 and 500 nM markedly reduced phosphorylation of AKT but not PI3K ⁇ 85 ⁇ / ⁇ and Src, following fMLP treatment (FIG. 1A). Treatment of patient neutrophils with 50 and 500 nM ARQ 092 significantly inhibited the surface amount of ⁇ 2 integrin following fMLP stimulation (FIG. 1 B). Further, binding of anti-activated ⁇ 2 antibodies (CBRM1/5) was decreased by treatment with ARQ 092 (FIG. 1C).
  • ARQ 092 inhibits the function of platelet surface molecules. It was found that treatment with 50 and 500 nM ARQ 092 inhibited AKT phosphorylation in thrombin-stimulated patient platelets without affecting PI3K and Src phosphorylation (FIG. 1 E). Treatment of patient platelets with 50 and 500 nM ARQ 092 significantly reduced P- selectin exposure, an indicator of a granule secretion, after stimulation with 0.025 U/ml thrombin (FIG. 1 F). As seen in neutrophils, while 5 nM ARQ 092 exhibited a significant but not complete inhibition of AKT phosphorylation, little inhibitory effects were observed in P-selectin exposure (data not shown).
  • ARQ 092 attenuates heterotypic aggregation of patient neutrophils and platelets under stirring conditions.
  • Heterotypic cell-cell interactions can cause vaso-occlusion in SCD patients. 13 Since ARQ 092 inhibited the function of surface molecules in neutrophils and platelets isolated from SCD patients, it was investigated whether ARQ 092 affects heterotypic cell-cell aggregation in vitro. Neutrophils and platelets isolated from SCD patients aggregated under stirring conditions mimicking venous shear, creating a new cell population (R1 gate) in which most cells were positive for both L-selectin (a leukocyte marker) and CD41 a (allb, a platelet marker) (FIG. 2A).
  • L-selectin a leukocyte marker
  • CD41 a allb, a platelet marker
  • ARQ 092 specifically inhibits AKT phosphorylation in neutrophils and platelets and reduces cell activation in SCD mice ex vivo.
  • ARQ 092 As a control, ARQ 092 at 100 mg/kg did not alter the number of circulating blood cells in WT mice (Table 1) and had no effect on splenomegaly in SCD mice (data not shown). It was observed that phosphorylation of AKT but not Src and PI3K was disrupted in fMLP-stimulated neutrophils isolated from SCD mice treated with ARQ 092, compared with vehicle (FIG. 3A). The membrane translocation of ⁇ 2 integrin and soluble FG binding were significantly impaired in neutrophils from ARQ 092-treated mice (FIGS. 3B-3C).
  • Oral administration of HU and ARQ 092 reduces cell-cell interactions in the cremaster venules of TNF-a-challenged SCD mice.
  • ARQ 092 vehicle or the compound was given orally 2.5 hours after TNF-a injection, followed by surgical procedures (FIG. 4A). No spontaneous bleeding was observed at the surgery site in all mice.
  • fluorescence intensities of anti-CD42c antibodies we measured.
  • E-selectin and intercellular adhesion molecule-1 (ICAM-1) expressed on activated ECs are required for neutrophil rolling and adhesion, respectively, during vascular inflammation.
  • ICAM-1 expression was significantly decreased in mice treated with HU, ARQ 092, or both inhibitors, compared to each vehicle control (FIGS. 4J-4K).
  • Co-administration of HU and ARQ 092 efficiently blocks neutrophil transmigration into the alveoli of TNF-a-challenged SCD mice. Neutrophils rapidly transmigrate from the pulmonary microvasculature and cause lung injury during inflammation. It was observed that oral administration of HU or ARQ 092 significantly blocked neutrophil transmigration into the alveoli of TNF-a-challenged SCD mice (FIGS. 4L-4M). Co-administration of HU and ARQ 092, compared to HU or ARQ 092 alone, further decreased the number of transmigrated neutrophils. These results suggest that HU with AKT inhibition efficiently reduces lung inflammation in TNF-a-challenged SCD mice.
  • Co-administration of HU and ARQ 092 efficiently blocks numerous functions of neutrophils and platelets isolated from TNF-a-challenged SCD mice ex vivo.
  • Extracellular H2O2 generation was weakly decreased by HU or ARQ 092 but significantly impaired by both inhibitors (FIGS. 6H-6I). Although the precise mechanism(s) by which HU inhibits platelet functions remains to be determined, the results herein can demonstrate that the combination therapy of HU and ARQ 092 efficiently inhibits numerous platelet functions in TNF-a-challenged SCD mice.
  • HU alone had a small effect on the function of ⁇ 2 integrin, and PTIO treatment nullified the effect of HU, but not ARQ 092, on ⁇ 2 membrane translocation and FG binding (FIGS. 7A-7B).
  • the potentiated inhibitory effect of both inhibitors was restored with PTIO treatment to a level similar to the inhibitory effect of ARQ 092 alone. It was also observed that administration of PTIO reversed the inhibitory effect of HU, but not ARQ 092, on P-selectin exposure in activated platelets (FIG. 7C).
  • ARQ 092 is an orally-available, selective AKT inhibitor which is currently in Phase lb clinical trials for the treatment of certain cancers. 28
  • This Example can at least demonstrate that ARQ 092 reduces the adhesive function of neutrophils and platelets isolated from SCD patients in vitro. Oral administration of a single dose of ARQ 092 abrogated AKT phosphorylation in isolated neutrophils and platelets following agonist stimulation, was observed to significantly reduced cell-cell interactions in cremaster venules, and decreased neutrophil transmigration into the alveoli of TNF-a-challenged SCD mice. The inhibitory effects and survival were observed to be increased when the mice were pretreated with HU.
  • this Example can at least demonstrate that a specific inhibitor of AKT may be beneficial to treat acute vaso-occlusive events in SCD patients. Since AKT signaling is important for the function of intravascular cells during numerous vascular diseases, 12 ' 17-21 ' 23 ARQ 092 is likely to inhibit the activity of all AKT isoforms in intravascular cells and thereby attenuate the process of thrombosis and inflammation in SCD patients.
  • This Example can demonstrate that ARQ 092 reduces the ligand-binding function of ⁇ 2 integrin in stimulated neutrophils isolated from SCD patients. It was observed that ARQ 092 inhibits ⁇ 2 integrin function in neutrophils isolated from SCD mice after oral administration of the inhibitor.
  • this Example can at least demonstrate that co- administration of HU and ARQ 092 can inhibit cell-cell interactions during vaso-occlusion in SCD. Most AKT inhibitors including ARQ 092 are being developed as an anti-cancer drug.
  • this Example can also at least demonstrate that ARQ 092 can be an effective drug to treat and/or prevent thrombotic complications in a subject in need thereof, which in some instances can have a cancer and/or SCD.
  • This Example can at least demonstrate that oral administration of a single dose of both HU and ARQ 092 can efficiently inhibit neutrophil-EC and neutrophil- platelet interactions in cremaster venules and reduces neutrophil recruitment into the alveoli of TNF-a-challenged SCD mice. Survival was improved by HU alone or both HU and ARQ 092, but not ARQ 092 alone. It was reported that iv infusion of an AKT2 inhibitor decreases E- selectin and ICAM-1 expression on cremaster vessels of TNF-a-challenged SCD mice.
  • Oxidative stress can result in reduced NO bioavailability in SCD patients and mice, which in turn aggravates inflammatory conditions. 38 39 HU has many beneficial effects in SCD patients. Importantly, preclinical and clinical studies demonstrated that HU increases the plasma level of NO species and stimulates a cGMP-signaling pathway. 5 ' 6 8 9 As seen in SCD mice after iv infusion of HU, 9 oral administration of HU into SCD mice also increases the plasma NOx level.
  • ARQ 092 may inhibit EC AKT1-eNOS signaling
  • the plasma NOx level was not changed by ARQ 092 treatment alone in TNF-a-challenged SCD mice, implying that eNOS-derived NO production may not be important for maintaining the basal level of plasma NOx in the SCD mice.
  • an NO scavenger, PTIO does not alter the inhibitory effect of ARQ 092 on platelet and neutrophil function (FIGS. 7A-7C) can suggest that ARQ 092 is unlikely to affect cellular NO generation.
  • ARQ 092 significantly inhibits cell-cell interactions in microvessels of TNF-a-challenged SCD mice.
  • ARQ 092 showed synergistic effects on acute vaso-occlusive events and improved survival in the mice.
  • this Example can at least demonstrate that 50- 500 nM ARQ 092 can be efficacious in inhibiting platelet and neutrophil functions.
  • the plasma concentration of ARQ 092 reached 2.6, 6.4, and 8.1 ⁇ after oral administration of 20, 40, and 60 mg per day, respectively, for 15 days (unpublished data).
  • a minimal oral dose of ARQ 092 may be sufficient to attenuate vaso-occlusive events in SCD patients.
  • This Example can at least demonstrate that in addition to anti-cancer therapy, ARQ 092 can be a a treatment of acute vaso-occlusive episodes in SCD patients. This Example can also at least demonstrate that the combination of ARQ 092 and HU can be a treatment of acute vaso-occlusive episodes in SCD patients.
  • Hydroxyurea N-formyl-methionyl-leucyl-phenylalanine (fMLP), 2', 7'- dichlorofluorescein diacetate (DCFH-DA), human thrombin, and PGE1 were purchased from Sigma (St. Louis, MO).
  • D-Phe-Pro-Arg-chloromethyl ketone (PPACK) and a mouse anti-human CD42b (Glycoprotein Iba (GPIba), MM2/174) antibody were from EMD Millipore (Billerica, MA).
  • Collagen-related peptide (CRP) was obtained from Dr. Richard Farndale (Department of Biochemistry, University of Cambridge, UK).
  • Antibodies against p-PI3K ⁇ 85 ⁇ / ⁇ at Tyr458/p55a/v at Tyr199, p-Src at Tyr416, p-AKT at Ser473, and AKT were from Cell Signaling (Danvers, MA).
  • Recombinant mouse TNF-a an Alexa Fluor 647- conjugated antibody against mouse Ly-6G, a PE-conjugated antibody against mouse ⁇ 2 (M1/70), an APC-conjugated antibody against mouse PECAM-1 , an antibody against mouse ICAM- 1 , a Dylight 488-conjugated antibody against rat IgG, PE- conjugated monoclonal antibodies against total (ICRF44) or activated human ⁇ 2 (CBRM1/5), a Dylight 488-conjugated antibody against mouse IgG, and isotype control IgGs were purchased from BioLegend (San Diego, CA).
  • Unlabeled or FITC-conjugated control IgG or anti-human L-selectin antibodies were obtained from eBioscience (San Diego, CA).
  • DyLight 488- conjugated anti-mouse CD42c ( ⁇ ) and FITC- conjugated anti-mouse P-selectin (Wug.E9) or anti-CD42b (GPIba, Xia.B2) antibodies were purchased from Emfret Analytics (Eibelstadt, Germany).
  • Amplex® Red Hydrogen Peroxide/Peroxidase Assay Kit, CellTrackerTM Red CMTPX, Calcium AM, and DyLight 488 dye were obtained from Life Technologies (Grand Island, NY).
  • Human fibrinogen and human von Willebrand Factor (vWF) was purchased from Haematologic Technologies
  • Ca 2+ dye FLIPR Calcium Assay kit
  • Carboxy- PTIO (2-phenyl-4,4,5,5- tetramethylimidazoline-1-oxyl-3-oxide (potassium salt)) was obtained from Santa Cruz Biotechnology (Dallas, TX).
  • a Nitrate/Nitrite Colorimetric Assay kit was purchased from Cayman Chemical (Ann Arbor, Ml).
  • Chimeric Berkeley mice were generated by transplantation of bone marrow cells isolated from Berkeley mice into lethally irradiated WT mice. Briefly, bone marrow cells of Berkeley mice (8-12 weeks old) were harvested from both femurs and tibias as described previously. 2 After lysing red blood cells, marrow cells were washed with ice-cold RPMI 1640 containing 10 U/mL heparin and 20 mM HEPES, pH
  • mice containing 0.1 mg/mL neomycin 1 week before irradiation.
  • the recipient mice underwent radiation (950 rad) one day prior to receiving 5 x 10 6 bone marrow cells in 250 ⁇ RPMI 1640 by tail-vein injection.
  • Recipient mice were fed with sterile food and acidic water containing neomycin until the experiment.
  • age- matched (20- to 24-week old) chimeric Berkeley mice were used in our studies.
  • PCR and electrophoresis analyses were performed using blood from SCD mice.
  • 200 ⁇ of blood was treated with 1 ml red blood cell lysis buffer (8.3 mg/mL NH4CI, 1 mg/mL NaHC03, 0.01 mg/mL EDTA) at room temperature for 5 minutes. After spinning at 750 g for 5 minutes, the supernatant containing Hb was saved for electrophoresis.
  • the cell pellet was washed once with PBS and resuspended in 100 ⁇ Tris buffer (0.67 mM Tris-HCI, 0.07 mM EDTA pH 7.5).
  • Primers for detection of mouse Hb ⁇ (291 base pairs) are: forward, 5'- AGATGTTTTTTTCACATTCTTGAGC-3' (SEQ ID NO: 3), and reverse, 5'- AATGCCTGCTCTTTACTGAAGG-3' (SEQ ID NO: 4).
  • Primers for detection of human sickle transgene of Hb ⁇ (480 base pairs): forward, 5'-GTATGGGAGAGGCTCCAACTC-3'(SEQ ID NO: 5), and reverse, 5'-TCTGCCCAAATCTTAGACAAA AC-3'(SEQ ID NO: 6).
  • electrophoresis in a 15% acidic polyacrylamide gel containing urea was carried out by a modified method of Alter and Goff. 4
  • the gel solution consisted of 15% poly acrylamide (37.5:1 , acrylamide: bis-acrylamide), 5% glacial acetic acid, 4.25 M Urea which was freshly deionized with mixed bed resin (Sigma), 0.5% ammonium persulfate, and 3% TEMED.
  • the electrophoresis buffer was 5% acetic acid.
  • the gel was pre-electrophoresed twice with fresh running buffer for 60 minutes at 200 V with polarity reversed. The current fell from 30 mA to 10 mA during pre- electrophoresis, which indicated completion.
  • Hb, 2 ⁇ g, obtained from WT, Hbb+/-, and SCD (Hbb-/-) mice was mixed with 20 ⁇ sample buffer (8 M freshly deionized urea containing 5% acetic acid), followed by electrophoresis for 4 hours 15 minutes and stained with Coomassie blue. The degree of chimerism was determined by comparison of the band density of mouse and human Hba/ ⁇ .
  • mice were then treated with saline or 250 mg/kg of HU (50 mg/ml) by oral gavage and subsequently with ip injection of TNF- a (500 ng).
  • Phosphoric acid (0.01 M) or ARQ 092 (100 mg/kg) was administered orally at 2.5 hours after TNF-a treatment.
  • Blood and femur/tibia were collected at 3 hours after TNF-a treatment to isolate platelets and neutrophils, respectively.
  • Neutrophils were treated with or without fMLP (0.5 ⁇ for human and 10 ⁇ for mouse) or 20 ng/ml TNF- a for 10 minutes at 37°C, followed by incubation with PE-conjugated isotype control IgG or antibodies against human ⁇ 2 (ICRF44), activated human ⁇ 2 (CBRM1/5), or mouse ⁇ 2, or Alexa Fluor 488- conjugated FG. Platelets were incubated with or without 0.025 U/ml thrombin for 5 minutes at 37°C, followed by incubation with PE-conjugated control IgG or antibodies against human P-selectin for 15 minutes.
  • AKT phosphorylation in neutrophils and platelets in vitro and ex vivo Isolated neutrophils and platelets were pretreated with or without 0.1 % DMSO or ARQ 092, followed by stimulation with an agonist. Cell lysates were immunoblotted with antibodies against total or phospho-AKT (p-AKT).
  • p-AKT phospho-AKT
  • Berkeley mice were fasted overnight and treated with oral injection of vehicle or 100 mg/kg body weight (BW) of ARQ 092 (10 mg/ml in 0.01 M phosphoric acid) at 0.5 hours before collecting the blood and femur/tibia.
  • Isolated neutrophils or platelets were incubated with fMLP or thrombin, respectively, for 2 minutes at 37°C.
  • Equal amounts of proteins (50 ⁇ g) in cell lysates were immunoblotted with antibodies against p-AKT at Ser473, AKT, p-phosphoinositide 3- kinase (PI3K) ⁇ 85 ⁇ / ⁇ at Tyr458/p55a/v at Tyr199, or p-Src at Tyr416, followed by densitometry.
  • p-AKT Ser473, AKT, p-phosphoinositide 3- kinase (PI3K) ⁇ 85 ⁇ / ⁇ at Tyr458/p55a/v at Tyr199, or p-Src at Tyr416, followed by densitometry.
  • PI3K p-phosphoinositide 3- kinase
  • Neutrophil-platelet aggregation assay The in vitro cell-cell aggregation assay was performed under stirring conditions mimicking venous shear as we described. 5 Neutrophils and/or platelets isolated from SCD patients were incubated with 500 nM ARQ 092 for 30 minutes at 37°C and washed, followed by labeling with FITC-conjugated anti- L-selectin (2 ⁇ g/ml) and APC-conjugated anti-human CD41 a antibodies (15 ng/ml), respectively. Platelets were then treated with 0.025 U/ml thrombin at 37°C for 5 minutes.
  • activated platelets were mixed with neutrophils for 5 minutes under a stirring condition of 1 ,000 rpm in an aggregometer. Cells were fixed with 1 % paraformaldehyde and analyzed by flow cytometry as described above.
  • PPACK D-phenylalanyl-L-prolyl-L-arginine chloromethyl ketone
  • Platelet aggregation and agglutination assay Washed patient platelets were pre- treated with vehicle (0.01 % DMSO) or 50 and 500 nM ARQ 092 for 30 minutes at 37°C. Platelet aggregation was induced by stimulation with different concentrations of thrombin or CRP. For ex vivo studies, vehicle, HU, ARQ 092, or both were given orally to non- challenged or TNF-a-challenged Berkeley mice as described in flow cytometry. Isolated platelets were stimulated with thrombin or CRP. Platelet aggregation was measured in a platelet lumi-aggregometer (Chronolog Corp, Havertown, PA) at 37°C with stirring (1 ,000 rpm).
  • HEPES-Tyrode buffer (20 mM HEPES, pH 7.3, 136 mM NaCI, 2.7 mM KCI, 12 mM NaHCC , 1 mM MgCI 2 , and 5.5 mM glucose) without CaCI 2 and BSA.
  • Human platelets were incubated with 0.5 ⁇ g/ml human vWF and then 0.3 mg/ml ristocetin, and mouse platelets were treated with 10 ⁇ g/ml human vWF and then 10 ⁇ g/ml botrocetin, followed by measurement of agglutination in an aggregometer.
  • Plasma Nitrate/Nitrite Levels After recording survival times, blood was immediately drawn, and the plasma was isolated and stored at -80 °C until use. Plasma, 40 ⁇ , was used for a Nitrate/Nitrite Colorimetric Assay kit and the absorbance of each sample was measured at 540 nm using a microplate reader (PHERAstar, BMG Labtech).
  • Intracellular ROS generation was measured as described previously. 5 Vehicle, HU, ARQ 092, or both were given orally to TNF-a- challenged Berkeley mice as described in flow cytometry. Mouse neutrophils (2.5 x 10 5 ) and platelets (3 x 10 6 ) were incubated with 5 ⁇ DCFH-DA for 10 minutes at 37°C. After stimulation with 10 ⁇ fMLP or 0.025 U/ml thrombin, the DCF signal was measured by flow cytometry. The extracellular H2O2 level was measured using the Amplex® Red H2O2 assay kit (Thermo Fisher Scientific), according to the manufacturer's instructions.
  • neutrophils 5 x 10 5 in 50 ⁇ HBSS
  • platelets 5 x 10 7 in 50 ⁇ HEPES-Tyrode buffer containing 1 mM CaC .
  • Neutrophils and platelets were treated with or without fMLP and thrombin, respectively, and then mixed with 50 ⁇ of Amplex red reagent.
  • the Amplex red signal was measured immediately using a PHERAstar FS (BMG Labtech, Cary, NC) with an absorbance of 560 nm for 30 minutes.
  • Ca 2+ mobilization Vehicle, HU, ARQ 092, or both were administered orally to TNF-a- challenged Berkeley mice as described in flow cytometry.
  • Cells were incubated with Ca 2+ dye for 30 minutes at 37°C in the dark, and neutrophils and platelets were then treated with 10 ⁇ fMLP or 0.025 U/ml thrombin, respectively.
  • 2 mM CaC was added at 200- 300 seconds after treatment with an agonist.
  • the Ca 2+ signal was measured using a FlexStation spectrofluorometer (Molecular Devices) with an excitation wavelength of 485 nm and an emission wavelength of 525 nm.
  • the cytosolic Ca 2+ level was expressed as relative fluorescence unit and quantified by the area under the curve.

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  • Acyclic And Carbocyclic Compounds In Medicinal Compositions (AREA)

Abstract

L'invention concerne des procédés de traitement de la drépanocytose ou d'un symptôme de cette maladie chez un sujet en ayant besoin, ces procédés pouvant comprendre l'administration d'une quantité d'ARQ 092 ou d'une formulation pharmaceutique de cette substance au sujet en ayant besoin. Le procédé peut comprendre également l'administration d'une quantité d'hydroxyurée ou d'une formulation pharmaceutique d'hydroxyurée au sujet en ayant besoin.
PCT/US2018/054676 2017-10-05 2018-10-05 Procédé de traitement de la drépanocytose WO2019071171A1 (fr)

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Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20160067260A1 (en) * 2014-09-05 2016-03-10 Arqule, Inc. Compositions and Methods for Treating Proliferation Disorders

Patent Citations (1)

* Cited by examiner, † Cited by third party
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US20160067260A1 (en) * 2014-09-05 2016-03-10 Arqule, Inc. Compositions and Methods for Treating Proliferation Disorders

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