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WO2011084514A2 - Procédés et compositions pour traiter une complication associée à la malaria - Google Patents

Procédés et compositions pour traiter une complication associée à la malaria Download PDF

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Publication number
WO2011084514A2
WO2011084514A2 PCT/US2010/060565 US2010060565W WO2011084514A2 WO 2011084514 A2 WO2011084514 A2 WO 2011084514A2 US 2010060565 W US2010060565 W US 2010060565W WO 2011084514 A2 WO2011084514 A2 WO 2011084514A2
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Prior art keywords
sur1
fold
trpm4
antagonist
malaria
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WO2011084514A9 (fr
Inventor
J. Marc Simard
Vladimir V. Gerzanich
Gary Schwartzbauer
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University of Maryland Baltimore
University of Maryland College Park
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University of Maryland Baltimore
University of Maryland College Park
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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
    • A61K31/16—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
    • 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
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/445—Non condensed piperidines, e.g. piperocaine
    • A61K31/4523—Non condensed piperidines, e.g. piperocaine containing further heterocyclic ring systems
    • A61K31/454—Non condensed piperidines, e.g. piperocaine containing further heterocyclic ring systems containing a five-membered ring with nitrogen as a ring hetero atom, e.g. pimozide, domperidone
    • 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
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/455—Nicotinic acids, e.g. niacin; Derivatives thereof, e.g. esters, amides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P33/00—Antiparasitic agents
    • A61P33/02—Antiprotozoals, e.g. for leishmaniasis, trichomoniasis, toxoplasmosis
    • A61P33/06—Antimalarials
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the invention relates to methods and compositions for treating a malaria- related complication.
  • the invention further relates to kits comprising agents for a treating malaria-related complication.
  • CM 'cerebral malaria'
  • CM has features of diffuse encephalopathy, similar to that seen in metabolic conditions. Most patients have a history of fever, rigors, headache, vomiting, or seizures. Altered sensorium may present from the outset, or may develop over a period of days. The patient gradually becomes lethargic and lapses into coma (common in adults) or may become unconscious following a seizure (usually in children). Signs of irritability, restlessness or psychotic behavior may be initial manifestations of cerebral involvement Other signs and symptoms of CM include, impaired consciousness with non-specific fever; generalized convulsions and neurological sequelae; coma; and death.
  • the invention relates to the finding that the SUR1/TRPM4 channel (also known as the SUR1 -regulated non-selective cation calcium- ATP channel (NCc » . ATP channel)) contributes to the pathophysiology of malaria infection.
  • the SUR1/TRPM4 channel contributes to a malaria-related complication.
  • a method of the invention is drawn to treating a malaria-related complication.
  • a method of treating a malaria-related complication comprises administering a therapeutically effective amount of an antagonist of the SUR1/TRPM4 channel to a subject in need thereof.
  • the malaria-related complication is cerebral malaria (CM).
  • a method of the invention further comprises administering another agent or treatment used to treat CM in combination with an antagonist of the SUR1/TRPM4 channel.
  • a method of the invention is drawn to diagnosis of a malaria-related complication.
  • the method of diagnosis comprises comparative analysis of SUR1/TRPM4 expression level in a test sample.
  • the malaria-related complication is CM.
  • FIG. 1 Immunoblot for SUR1 of brain tissue from mice infected with P. Berghei ANKA parasites. Samples were taken from a mouse with systemic malaria but no cerebral involvement (M) and from another mouse with systemic malaria that was moribound, a cardinal manifestation of cerebral malaria (CM). Note the significant increase in SURl in the brain of the mouse with CM; the panel on the left shows molecular weight markers.
  • FIG. 2 Upregulation of Surl in malaria patients.
  • A, C Blood vessels from the brain of a patient infected with malaria, (arrows indicate the presence of Plasmodium parasites).
  • B Abundant expression of Surl in tissues.
  • D Surl was upregulated in microvascular endothelium (arrowheads) and in neurons (asterisks) and was localized to the parasites themselves (arrows).
  • the term “about” refers to a numeric value, including, for example, whole numbers, fractions, and percentages, whether or not explicitly indicated.
  • the term “about” generally refers to a range of numerical values (e.g., +/- 5-10% of the recited value) that one of ordinary skill in the art would consider equivalent to the recited value (e.g., having the same function or result).
  • the term “about” may include numerical values that are rounded to the nearest significant figure.
  • the term "antagonist,” “inhibitor,” or “blocker” and their grammatical variants may be used interchangeably and each refers to the ability to block, partially block, interfere, decrease, reduce or deactivate a channel such as the SUR1/TRMP4 channel.
  • antagonize, inhibit, or block encompasses a complete and/or partial loss of activity of a channel, such as the SUR1/TRMP4 channel.
  • channel activity may be inhibited by channel block (occlusion or closure of the pore region, preventing ionic current flow through the channel), by changes in an evening rate or in the mean open time, changes in a closing rate or in the mean closed time, or by other means.
  • a complete and/or partial loss of activity of the SUR1/TRMP4 channel as may be indicated by a reduction in cell depolarization, reduction in sodium ion influx or any other monovalent ion influx, reduction in an influx of water, reduction in extravasation of blood, reduction in cell death, as well as an improvement in cellular survival as a result of, for example, CM.
  • the term "compound” or “agent” is a molecular entity including, for example, a small molecule (especially small organic molecules that satisfy the constraints of Lipinski's Rules (Lipinski, C.A. et aL (1997) “Experimental And Computational Approaches To Estimate Solubility And Permeability In Drug Discovery And Development Settings," Adv. Drug Del. Rev, 23:3-25; Lipinski, C.A. et al. (2001) “Experimental And Computational Approaches To Estimate Solubility And Permeability In Drug Discovery And Development Settings," Adv. Drug Del. Rev. 46,3-26; Oprea, T.I. et al.
  • nucleic acid e.g., an oligonucleotide, and in particular, a siRNA, a shRNA an expression cassette, an antisense DNA, an and sense RNA, etc.
  • protein, peptide, antibody, antisense drug, or other biomolecule that is naturally made, synthetically made, or semi-synthetically made and is used alone or in combination with other therapies or methods for the stated purposes herein.
  • the terms "effective amount” or “therapeutically effective amount” in relation to compositions to treat a pathological condition of the invention are interchangeable and refer to an amount that results in an improvement, amelioration, or remediation of the symptoms of the disease or condition. Those of skill in the art understand that the effective amount may improve the patient's or subject's condition, but may not be a complete cure of the disease and/or condition. As used herein, the terms “effective amount” or “therapeutically effective amount” in relation to agents used to treat CM refers to an amount that results in improving a sign or symptom of CM.
  • a malaria-related complication includes signs and symptoms of malaria infection.
  • a malaria-related complication includes moderate to severe shaking, chills, fever, moderate to profuse sweating, general feeling of unease and discomfort (malaise), body aches, headache, nausea, vomiting, diarrhea, destruction of red blood cells, anemia, jaundice (yellowing of the skin or whites of the eyes), enlarged spleen, enlarged liver, increased respiratory rate, CM, and death.
  • a malaria- related condition is CM or death.
  • a malaria-related condition is CM.
  • sample refers typically to any type of material of biological origin that comprises, consists essentially of, or consists of, for example, a nucleic acid, protein, antibody, lipid, organelle, cell, intracellular or extracellular fluid, tissue, or an organ or isolated organ from a subject, including, for example, DNA, RNA, mitochondrion, nuclei, blood, plasma, serum, fecal matter, urine, semen, bone marrow, bile, spinal fluid, lymph fluid, samples of the skin, external secretions of the skin, respiratory, intestinal, genitourinary tracts, and other internal cavities, tears, saliva, milk, blood cell, muscle, organ, or other material of biological origin known by those of ordinary skill in the art
  • a sample is brain or other central nervous system tissue.
  • a sample is brain or other central nervous system tissue.
  • a sample may be cerebrospinal fluid (which may be obtained, for example, by lumbar or cervical puncture or from a cathether inserted into the ventricular system), or a brain tissue sample (which may be obtained, for example, by biopsy of the cortex).
  • cerebrospinal fluid which may be obtained, for example, by lumbar or cervical puncture or from a cathether inserted into the ventricular system
  • a brain tissue sample which may be obtained, for example, by biopsy of the cortex.
  • the sample or biological sample comprises, consists essentially of, or consists of the particular sample recited.
  • a sample from brain or other central nervous system tissue, cerebrospinal fluid, or other origin or composition described herein includes a sample comprising, consisting essentially of, or consisting of a sample from brain or other central nervous system tissue, cerebrospinal fluid, or other origin or composition of sample described herein.
  • a reference sample refers to a sample that is compared to a sample to determine the diagnosis, monitoring, or prognosis aspect of the invention described herein including, for example, in the case of diagnosis a sample that is known not to be infected by a malaria causing parasite or in the case of monitoring or prognosis a sample that provides a baseline measure to be used for monitoring or prognosis.
  • a sample can include material of biological origin that has been physically isolated from a subject and can also include material of biological origin that has not been physically isolated from a subject
  • a sample that has not been physically isolated can be analyzed using in vivo imaging/detection techniques.
  • SUR1 antagonist As used herein, the terms "SUR1 antagonist,” “SUR1 inhibitor,” and “SUR1 blocker” and their grammatical variants may be used interchangeably and each refers to compounds that reduce the activity or effect of a SUR1 receptor or functional unit, and include (but are not limited to) such compounds as, for example, glibenclarnide (also known as glyburide), tolbutamide, repaglinide, nateglinide, meglitinide, midaglizole, LY397364, LY389382, glyclazide, glimepiride, estrogen, estrogen related-compounds (estradiol, estrone, estriol, genistein, non-steroidal estrogen (e.g., diethystilbestrol), phytoestrogen (e.g., coumestrol), zearalenone, etc.) and combinations thereof.
  • glibenclarnide also known as glyburide
  • tolbutamide tolbut
  • Chemical names of some SUR1 antagonists include: glibenclarnide (l(p-2[S-chloro-0-anisamido)ethyl] phenyl] sulfonyl]-3-cyclohexyl-3-urea); chlopropamide (l-[[(p- chlorophenyl)sulfonyl]-3-propylurea glipizide (l-cyclohexyl-3[[p-[2(5- methylpyrazine carboxamido) ethyl] phenyl] sulfbnyl] urea); and tolazamide (berizeriesulfonamide-N-[[(hexahydro-lH-azepm-lyl)amino] carbonyl]-4-methyl).
  • TRPM4 antagonist As used herein, the terms "TRPM4 antagonist,” ' RPM4 inhibitor,” and “TRPM4 blocker” and their grammatical variants may be used interchangeably and each refers to compounds that reduce the activity or effect of a TRPM4 pore or functional unit, e.g.
  • ions by reducing or blocking the flow of ions through the TRPM4 pore, and include (but are not limited to) such compounds as, for example, pinkolant, rimonabant, riluzole, a fenamate (such as flufenamic acid, mefenamic acid, meclofenamic acid, or niflumic acid), l-(beta-[3-(4-methoxy- phenyl)propoxy]- 4-methoxyphenethyl)-lH-imidazole hydrochloride, and biologically active derivatives thereof.
  • a fenamate such as flufenamic acid, mefenamic acid, meclofenamic acid, or niflumic acid
  • l-(beta-[3-(4-methoxy- phenyl)propoxy]- 4-methoxyphenethyl)-lH-imidazole hydrochloride and biologically active derivatives thereof.
  • treat and all its forms and tenses (including, for example, treating, treated, and treatment) can refer to therapeutic or prophylactic treatment.
  • those in need of treatment include those already with a pathological condition of the invention (including, for example, CM), in which case treating refers to administering to a subject (including, for example, a human or other mammal in need of treatment) a therapeutically effective amount of a composition so that the subject has an improvement in a sign or symptom of a pathological condition of the invention.
  • the improvement may be any observable or measurable improvement
  • a treatment may improve the patient's condition, but may not be a complete cure of the pathological condition.
  • those in need thereof of treatment include, those in which a pathological condition of the invention (including, for example, CM) is to be prevented, in which case treating refers to administering to a subject a therapeutically effective amount of a composition to a subject (including, for example, a human or other mammal in need of treatment) at risk of developing a pathological conditional of the invention.
  • a pathological condition of the invention including, for example, CM
  • treating refers to administering to a subject a therapeutically effective amount of a composition to a subject (including, for example, a human or other mammal in need of treatment) at risk of developing a pathological conditional of the invention.
  • the subject in which a pathological condition of the invention is to be prevented has been infected with a malarial parasite, or is suspected of being infected with a malarial parasite.
  • CM complex multi-gens
  • malarial parasites apparently remain intravascular.
  • Parasitized red blood cells express malarial antigens that act as a docking signal to allow binding to vascular endothelial cells, resulting in microvascular sequestration in various organs (MacPherson et al., Am. J. Pathol. 119, 385 (1985); Pongponratn et al., Am. J. Trap. Med. Hyg. 44, 168 (1991); Silamut et al., Am. J. Pathol.
  • Epo erythropoietin
  • the SUR1/TRPM4 channel is a non-selective cation channel and is a critical regulator of CNS damage following cerebral ischemic and inflammatory microvascular injury (Chen et al., J Neurosci. 2003 Sep 17;23(24):8568-77; Gerzanich et al., Nat Med. 2009 Feb; 15(2): 185-91).
  • the SUR1/TRPM4 channel is also known as the SUR1 -regulated non-selective cation calcium- ATP channel (NC Ca-ATP channel).
  • NC Ca-ATP channel non-selective cation calcium- ATP channel
  • mis non-selective cation channel is transcriptionally upregulated in, for example, endothelial cells, oligodendrocytes, astrocytes, and neurons.
  • SUR1/TRPM4 channel transcriptional control of the SUR1 subunit is mediated in part by hypoxia-inducible factor- 1 (HIF-1) and NF-kappa-B, factors that are also known to be upregulated during ischemia, trauma, and CM (Simard et al, J. Cereb. Blood Flow Metab 29, 317 (2009); Medana et al., Int J. Parasitol. 36, 555 (2006); Tripathi et al., Blood (2009), 16). Therefore, without being bound by theory, the inventors believe that an inhibitor of the SUR1/TRPM4 channel (or combination thereof) reduces injury associated with CM by inhibiting this channel.
  • SUR1/TRPM4 Channel hypoxia-inducible factor- 1
  • SUR1/TRPM4 was identified first in native reactive astrocytes (NRAs) and later at least in neurons and capillary endothelial cells after stroke or traumatic brain or spinal cord injury (see, e.g., International application WO 03/079987 to Simard et al., and Chen M, Simard J M. J. Neurosci. 2001; 21:6512-6521, each incorporated by reference herein in its entirety).
  • This ion channel is also known to be the SUR1 -regulated non-selective cation calcium- ATP channel (NCC J - A TP channel) described in, for example, U.S. Pat Appl. Pub. Nos. 20030215889 and 20060100183.
  • the SUR1/TRPM4 channel is expressed at least on neural cells, neuroglial cells, and/or neural endothelial cells as a result of CM.
  • the channel is activated by intracellular calcium and blocked by intracellular ATP, and can be expressed in neuronal cells, neuroglia cells (also termed glia, or glial cells, e.g., astrocyte, ependymal cell, oligodentrocyte and microglia) or neural endothelial cells (e.g., capillary endothelial cells) in which the cells have been or are exposed to a traumatic insult, for example, an acute neuronal insult (e.g., hypoxia, ischemia, tissue compression, mechanical distortion, cerebral edema or cell swelling), toxic compounds or metabolites, an acute injury, cancer, brain abscess, etc., for example.
  • an acute neuronal insult e.g., hypoxia, ischemia, tissue compression, mechanical distortion, cerebral edema or cell swelling
  • the SUR1/TRPM4 channel is activated by intracellular calcium ions (Ca 2+ ) and is blocked by intracellular ATP. When opened by depletion of intracellular ATP, this channel is responsible for complete depolarization due to massive Na + influx, which creates an electrical gradient for C1- and an osmotic gradient for H 2 O, resulting in cytotoxic edema and cell death. When the channel is blocked or inhibited, massive Na + influx does not occur, thereby preventing cytotoxic edema.
  • the SUR1/TRPM4 channel of the present invention has a single-channel conductance to potassium ion (K + ) between 20 and 50 pS.
  • the SUR1/TRPM4 channel is also stimulated by Ca 2+ on the cytoplasmic side of the cell membrane in a physiological concentration range, where concentration range is from 10 -8 to 10 -5 M.
  • the SUR1/TRPM4 channel is also inhibited by cytoplasmic ATP in a physiological concentration range, where the concentration range is from 10 -1 to 10 M.
  • the SUR1/TRPM4 channel is also permeable to the following cations; K + , Cs + , Li + , Na + ; to the extent that the permeability ratio between any two of the cations is greater than 0.5 and less than 2.
  • the channel is regulated by sulfonylurea receptor type 1 (SUR1), which was originally considered to be associated exclusively with KATP channels such as those found in pancreatic beta-cells.
  • SUR imparts sensitivity to antidiabetic sulfonylureas such as glibenclamide and tolbutamide, for example, and is responsible for activation by a chemically diverse group of agents termed "K + channel openers" such as diazoxide, pinacidil and cromakalin (Aguilar-Bryan L, et al., Science. 1995; 268:423-426; Inagaki N, et al., Neuron.
  • K + channel openers such as diazoxide, pinacidil and cromakalin
  • the KATP channel in pancreatic beta-cells is formed from SUR1 linked with Kir6.2, whereas the cardiac and smooth muscle KATP channels are formed from SUR2A and SUR2B linked with Kir6.2 and Kir6.1, respectively (Fujita A, et al., Pharmacol Ther. 2000; 85:39- 53).
  • the SUR1/TRPM4 channel is also sensitive to sulfonylurea compounds and other SUR1 antagonists.
  • the SUR1/TRPM4 channel conducts sodium ions, potassium ions, cesium ions and other monovalent cations with near equal facility (Chen M, Simard J M. J. Neurosci. 2001; 21 :6512-6521) suggesting further that the characterization, and consequently the affinity to certain compounds, of the SUR1/TRPM4 channel differs from the KATP channel.
  • Nonselective cation channels that are activated by intracellular Ca 2+ and inhibited by intracellular ATP have been identified by others, but not in astrocytes, neurons, or other neural tissue as disclosed herein. Further, the SUR1/TRPM4 channel expressed and found in astrocytes differs physiologically from the other channels with respect to calcium sensitivity and adenine nucleotide sensitivity (Chen M, Simard J M. J. Neurosci. 2001; 21:6512-6521).
  • the SUR1/TRPM4 channel includes at least a regulatory component (regulatory subunit) and a pore-forming component (pore- forming subunit).
  • the regulatory subunit includes sulfonylurea type 1 receptor (SUR1) and the pore- forming subunit includes a nonselective cation channel subunit that is, or closely resembles, a transient receptor potential melastatin 4 (TRPM4) pore.
  • pathological diseases and conditions may be treated or prevented by inhibition of the SUR1/TRPM4 channel (including, for example, CM).
  • the SUR1/TRPM4 channel may be inhibited by reducing its activity, by reducing the numbers of such channels present in cell membranes, and by other means.
  • the SUR1/TRPM4 channel may be inhibited by administration of SUR1 antagonists; by administration of TRPM4 antagonists; by administration of a combination of drugs including a SUR1 antagonist and a TRPM4 antagonist; by reducing or antagonizing the expression, transcription, or translation of genetic message encoding the SUR1/TRPM4 channel; by reducing or antagonizing the insertion of SUR1/TRPM4 channels into cell membranes; and by other means known to one of ordinary skill in the art
  • the SUR1/TRPM4 channel is regulated by sulfonylurea receptor 1 (SURl) (e.g., it is opened by ATP depletion).
  • SUR1- regulated channels have been shown to play an important role in cytotoxic edema, oncotic cell death, and hemorrhagic conversion in ischemic stroke and CNS trauma.
  • SURl is blocked by SURl antagonists such as, for example, glibenclamide and tolbutamide, providing an exemplary avenue for treatment of a disease of the invention (including, for example, CM).
  • TRPM4 pores may be blocked by TRPM4 antagonists (e.g., TRPM4 blockers such as, for example, pinkolant, rimonabant, or a fenamate).
  • TRPM4 antagonists e.g., TRPM4 blockers such as, for example, pinkolant, rimonabant, or a fenamate.
  • CM leads to transcriptional activation of SURl and increased activity of SUR1/TRPM4 channels, initiating a cascade of events culminating in acute cerebral injury associated with CM.
  • CM may be treated by inhibition of the SUR1/TRPM4 channel, e.g., by administration of a SURl antagonist, a TRPM4 antagonist, a combination of a SUR1 antagonist and a TRPM4 antagonist, or by other means described herein.
  • SUR1/TRPM4 Channel Inhibitors e.g., TRPM4 blockers such as, for example, pinkolant, rimonabant, or a fena
  • an antagonist of the SUR1/TRPM4 channel includes one or more compounds capable of one or more of the following: (1) blocking the channel; (2) preventing channel opening; (3) inhibiting the channel; (4) reducing the magnitude of membrane current through the channel; (5) inhibiting transcriptional expression of the channel; and/or (6) inhibiting post-translational assembly and/or trafficking of channel subunits.
  • the SUR1/TRPM4 channel can be inhibited by a SUR1/TRP 4 channel inhibitor, a type 1 sulfonylurea receptor (SUR1) antagonist, a TRPM4 inhibitor, or a compound capable of reducing the magnitude of membrane current through the channel, or a combination or mixture thereof.
  • the SUR1 inhibitor is a sulfonylurea compound or a benzamido derivative.
  • a SUR1 inhibitor such as iptakalim may be used.
  • an exemplary SUR1 antagonist may be selected from the group consisting of glibenclamide, tolbutamide, repaglinide, nateglinide, meglitinide, midaglizole, LY397364, LY389382, glyclazide, glimepiride, estrogen, estrogen related-compounds (estradiol, estrone, estriol, genistein, non-steroidal estrogen (e.g., diethystilbestrol), phytoestrogen (e.g., coumestrol, zearalenone, etc.), and compounds known to inhibit or block KATP channels.
  • MgADP can also be used to inhibit the channel.
  • KATP channels include, but are not limited to tolbutamide, glyburide (l[p-2[5-chloro-0-anisamido)ethyl] phenyl] sulfonyl]-3-cyclohexyl-3-urea); chlopropamide (l-[[(p-chlorophenyl) sulfonyl]-3-propylurea; glipizide (l-cyclohexyl-3 [ [p- [2(5-methylpyrazine carboxamido)ethyl] phenyl] sulfonyl] urea); or tolazamide(benzenesulfonamide-N- [[(hexahydro-1H-azepin-lyl)amino] carbonyl]-4-methyl).
  • the channel blocker is a TRPM4 channel inhibitor and is selected from the group consisting of pinkolant, rimonabant, riluzole, a fenamate (such as, for example, flufenamic acid, mefenamic acid, meclofenamic acid, or niflumic acid), l-(beta-[3-(4-methoxy- phenyl)propoxy]-4-methoxyphenethyl)-1H-imidazole hydrochloride, and a biologically active derivative thereof.
  • a TRPM4 channel inhibitor is selected from the group consisting of pinkolant, rimonabant, riluzole, a fenamate (such as, for example, flufenamic acid, mefenamic acid, meclofenamic acid, or niflumic acid), l-(beta-[3-(4-methoxy- phenyl)propoxy]-4-methoxypheneth
  • non-sulfonyl urea compounds such as 2, 3-butanedione and 5- hydroxydecanoic acid, quinine, and therapeutically equivalent salts and derivatives thereof, may be employed in the invention.
  • the benzamido derivative may be selected from the group consisting of repaglinide, nateglinide, and meglitinide.
  • the inhibitor may comprise a protein, a peptide, a nucleic acid (such as an RNAi molecule or antisense RNA, including siRNA), or a small molecule.
  • the inhibitor is provided intracranialy, intravenously, subcutaneously, intramuscularly, intracutaneously, intragastrically, or orally.
  • the inhibitor is provided intracranialy or intravenously.
  • the method further comprises administering MgADP to the individual.
  • An agent of the invention that inhibits the SUR1/TRPM4 channel for the treatment of a pathological condition of the invention may be delivered in any suitable manner, but in particular embodiments they are delivered intravenously, intra -arterially, intracranially, or intraperitoneally.
  • the composition(s) of the present invention may be delivered alimentarily or parenterally. Examples of alimentary administration include, but are not limited to, orally, buccally, rectally, or sublingually.
  • Parenteral administration can include, but is not limited to, intramuscularly, subcutaneously, intraperitoneally, intravenously, intratumorally, intraarterially, intraventricularly, intracavity, intravesical, intrathecal, or intrapleural.
  • Other modes of administration may also include topically, mucosally, transdermally, or by direct injection into the brain or other compartment of the CNS.
  • the therapeutically effective amounts of a compound for treating a pathological condition of the brain are those amounts effective to produce beneficial results, which may be initially detennined by one of ordinary skill in the art by reviewing the published literature, by conducting in vitro tests or by conducting metabolic studies in healthy experimental animals, for example. Before use in a clinical setting, it may be beneficial to conduct confirmatory studies in an animal model, preferably a widely accepted animal model of the particular disease to be treated.
  • Preferred animal models for use in certain embodiments are rodent models, which are preferred because they are economical to use and, particularly, because the results gained are widely accepted as predictive of clinical value.
  • a specific dose level of active compounds for any particular patient depends upon a variety of factors including the activity of the specific compound employed, the age, body weight, general health, sex, diet, time of administration, route of administration, rate of excretion, drug combination, and the type or severity of the particular disease undergoing therapy.
  • the person responsible for administration will determine the appropriate dose for the individual subject.
  • a dose is determined prior to administration of an agent of the invention for treating CM.
  • preparations should meet sterility, pyrogenicity, and general safety and purity standards.
  • the treatments may include various "unit doses.”
  • Unit dose is defined as containing a predetermined quantity of the therapeutic composition (e.g., an antagonist of the SUR1/TRPM4 channel or its related-compounds thereof) calculated to produce the desired responses in association with its administration (e.g., the appropriate route and treatment regimen).
  • the quantity to be administered, and the particular route and formulation, are within the skill of those in the clinical arts.
  • Also of import is the subject to be treated, in particular, the state of the subject and the protection desired.
  • a unit dose need not be administered as a single injection, but may comprise continuous infusion over a set period of time.
  • Systemic administration or oral administration may be performed, and, in embodiments of the present invention, local or regional administration may be performed.
  • Continuous administration also may be applied where appropriate, for example, where a patient may be monitored on an on-going basis. Delivery via syringe or catheterization is one effective method. Continuous infusion may take place for a period from about 1-2 hours, to about 2-6 hours, to about 6-12 hours, to about 12-24 hours, to about 1-2 days, to about 1-2 wk or longer following the initiation of treatment
  • the dose of the therapeutic composition via continuous infusion will be equivalent to that given by a single or multiple injections, adjusted over a period of time during which the infusion occurs. Multiple injections delivered as single dose comprise about 0.1 to about 1 ml volumes.
  • the volume to be administered may be about 4- 10 ml (preferably 10 ml), while in further embodiments a volume of about 1-3 ml will be used (preferably 3 ml).
  • compositions include pharmaceutical compositions comprising a therapeutically effective amount of one or more of the active compounds or substances along with a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable carrier means a non-toxic, inert solid, semi-solid liquid filler, diluent, encapsulating material, formulation auxiliary of any type, or simply a sterile aqueous medium, such as saline.
  • sugars such as lactose, glucose and sucrose, starches such as corn starch and potato starch, cellulose and its derivatives such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt, gelatin, talc; excipients such as cocoa butter and suppository waxes; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols, such as propylene glycol, polyols such as glycerin, sorbitol, mannitol and polyethylene glycol; esters such as ethyl oleate and ethyl laurate, agar, buffering agents such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water, isotonic saline, Ringer's solution; ethyl
  • wetting agents such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, releasing agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the composition, according to the judgment of the formulator.
  • antioxidants examples include, but are not limited to, water soluble antioxidants such as ascorbic acid, cysteine hydrochloride, sodium bisulfite, sodium metabisulfite, sodium sulfite, and the like; oil soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, aloha-tocopherol and the like; and the metal chelating agents such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid and the like.
  • water soluble antioxidants such as ascorbic acid, cysteine hydrochloride, sodium bisulfite, sodium metabisulfite, sodium sulfite, and the like
  • oil soluble antioxidants such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (B
  • Toxicity and therapeutic efficacy of such compounds can be determined by standard pharmaceutical procedures in cell assays or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population).
  • the dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD50 ED50.
  • Compounds which exhibit large therapeutic indices may be preferred. While compounds that exhibit toxic side effects may be used, care should be taken to design a delivery system that targets such compounds to the site of affected tissue in order to minimize potential damage to uninfected cells and, thereby, reduce side effects.
  • the data obtained from the cell culture assays and animal studies can be used in formulating a range of dosage for use in humans.
  • the dosage of such compounds he preferably within a range of circulating concentrations that include the ED50 with little or no toxicity.
  • the dosage may vary within this range depending upon the dosage form employed and the route of administration utilized.
  • the therapeutically effective dose can be estimated initially from cell based assays.
  • a dose may be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 (i.e., the concentration of the test compound which achieves a half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans.
  • an effective amount or therapeutically effective amount of an antagonist of the invention as a treatment varies depending upon the host treated and the particular mode of administration.
  • the dose range of the antagonist will be about 0.01 ⁇ g/kg body weight to about 20,000 ⁇ g/kg body weight
  • body weight is applicable when an animal is being treated.
  • a variety of different dosage levels will be of use, for example, 0.0001 ⁇ g/kg, 0.0002 ⁇ g/kg, 0.0003 ⁇ g/kg, 0.0004 ⁇ g/kg, 0.005 ⁇ g/kg, 0.0007 ⁇ g/kg, 0.001 ⁇ g/kg, 0.1 ⁇ g/kg, 1.0 ⁇ g/kg, 1.5 ⁇ g/kg, 2.0 ⁇ g/kg, 5.0 ⁇ g/kg, 10.0 ⁇ g/kg, 15.0 ⁇ g/kg, 30.0 ⁇ g/kg, 50 ⁇ g/kg, 75 ⁇ g/kg, 80 ⁇ g/kg, 90 ⁇ g/kg, 100 ⁇ g/kg, 120 ⁇ g/kg, 140 ⁇ g/kg, 150 ⁇ g/kg, 160 ⁇ g/kg, 180 ⁇ g/kg, 200 ⁇ g/kg, 225 ⁇ g/kg, 250 ⁇ g/kg, 275 ⁇ g/kg, 300 ⁇ g/kg,
  • the amount of the antagonist administered to the subject is in the range of about 0.0001 ⁇ g/kg/day to about 20 mg/kg/day, about 0.01 ⁇ g/kg/day to about 100 ⁇ g/kg/day, or about 100 ⁇ g/kg/day to about 20 mg/kg/day.
  • the antagonist may be administered to the subject in the form of a treatment in which the treatment may comprise the amount of the antagonist or the dose of the antagonist that is administered per day, week, month, etc.
  • Treatments may be administered such that the amount of antagonist administered to the subject is in the range of about 0.0001 ⁇ g/kg/treatment to about 20 mg/kg/treatment, about 0.01 ⁇ g/kg/treatment to about 100 ⁇ g/kg/treatment, or about 100 ⁇ g/kg/treatment to about 20 mg kg/treatment
  • the total daily dose of an active compound of the present invention administered to a subject in single or in divided doses can be in amounts, for example, from 0.01 to 25 mg/kg body weight or more usually from 0.1 to IS mg/kg body weight
  • Single dose compositions may contain such amounts or submultiples thereof to make up the daily dose.
  • treatment regimens according to the present invention comprise administration to a human or other mammal in need of such treatment from about 1 mg to about 1000 mg of the active substance(s) of this invention per day in multiple doses or in a single dose of from 1 mg, 5 mg, 10 mg, 100 mg, 500 mg or 1000 mg, in certain embodiments.
  • Liquid dosage forms for oral administration may include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs containing inert diluents commonly used in the art, such as water, isotonic solutions, or saline.
  • Such compositions may also comprise adjuvants, such as wetting agents; emulsifying and suspending agents; sweetening, flavoring and perfuming agents.
  • Injectable preparations for example, sterile injectable aqueous or oleaginous suspensions, may be formulated according to what is known in the art using suitable dispersing or wetting agents and suspending agents.
  • the sterile injectable preparation may also be a sterile injectable solution, suspension or emulsion in a nontoxic parenterally acceptable diluent or solvent, for example, as a solution in 1,3-butanediol.
  • the acceptable vehicles and solvents that may be employed are water, Ringer's solution, U.S.P. and isotonic sodium chloride solution.
  • sterile, fixed oils are conventionally employed as a solvent or suspending medium.
  • any bland fixed oil can be employed including synthetic mono- or diglycerides.
  • fatty acids such as oleic acid are used in the preparation of injectables.
  • the injectable formulation can be sterilized, for example, by filtration through a bacteria-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions, which can be dissolved or dispersed in sterile water or other sterile injectable medium just prior to use.
  • Solid dosage forms for oral administration may include capsules, tablets, pills, powders, gelcaps, and granules.
  • the active compound may be admixed with at least one inert diluent such as sucrose, lactose or starch.
  • Such dosage forms may also comprise, as is normal practice, additional substances other than inert diluents, e.g., tableting lubricants and other tableting aids such as magnesium stearate and microcrystalline cellulose.
  • the dosage forms may also comprise buffering agents. Tablets and pills can additionally be prepared with enteric coatings and other release-controlling coatings.
  • compositions of a similar type may also be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugar as well as high molecular weight polyethylene glycols and the like.
  • the active compounds can also be in micro-encapsulated form with one or more excipients as noted above.
  • the solid dosage forms of tablets, capsules, pills, and granules can be prepared with coatings and shells such as enteric coatings and other coatings well known in the pharmaceutical formulating art. They may optionally contain opacifying agents and can also be of a composition mat they release the active ingredient(s) only, or preferably, in a certain part of the intestinal tract, optionally in a delayed manner. Examples of embedding compositions which can be used include polymeric substances and waxes.
  • the method of the present invention employs the compounds identified herein for both in vitro and in vivo applications.
  • the invention compounds can be incorporated into a pharmaceutically acceptable formulation for administration.
  • suitable dosage levels refers to levels of compound sufficient to provide circulating concentrations high enough to effectively inhibit the SUR1/TRPM4 channel.
  • compositions comprising at least one SUR1 antagonist compound (as described above), at least one TRPM4 antagonist (as described above), or a combination thereof, and a pharmaceutically acceptable carrier are contemplated.
  • Exemplary pharmaceutically acceptable carriers include carriers suitable for oral, intravenous, subcutaneous, intramuscular, intracutaneous, and the like administration. Administration in the form of creams, lotions, tablets, dispersible powders, granules, syrups, elixirs, sterile aqueous or non-aqueous solutions, suspensions or emulsions, and the like, is contemplated.
  • suitable carriers include emulsions, solutions, suspensions, syrups, and the like, optionally containing additives such as wetting agents, emulsifying and suspending agents, sweetening, flavoring and perfuming agents, and the like.
  • suitable carriers include sterile aqueous or non-aqueous solutions, suspensions, or emulsions.
  • suitable carriers include sterile aqueous or non-aqueous solutions, suspensions, or emulsions.
  • non-aqueous solvents or vehicles are propylene glycol, polyethylene glycol, vegetable oils, such as olive oil and corn oil, gelatin, and injectable organic esters such as ethyl oleate.
  • Such dosage forms may also contain adjuvants such as preserving, wetting, emulsifying, and dispersing agents. They may be sterilized, for example, by filtration through a bacteria-retaining filter, by incorporating sterilizing agents into the compositions, by irradiating the compositions, or by heating the compositions.
  • the compounds of the present invention may be administered alone or in combination with another agent or treatment that is used to treat CM.
  • the compounds of the present invention used in combination with another agent or treatment can be administered prior to, concurrent with, or following the other agent or treatment
  • Chemotherapy drugs used for CM now primarily involves the use of quinine, for a patient with severe CM it may be assumed that chloroquine resistance is an issue.
  • Quinine is one of the four main alkaloids found in the bark of the Cinchona tree and is the only drug which over a long period of time has remained largely effective for treating the disease.
  • Quinine has similar activity to chloroquine in that it is likely to interfere with the parasite's enzymatic digestion.
  • Artemisinins have been shown in some clinical trials to clear parasitemia and fever fester than quinine or chloroquine, but they had no effect on mortality rates. Artemisinin has been used by the Chinese as a traditional treatment for fever and malaria. It is a sesquiterpene lactone derived from Artemisia annua. The two most widely used are artesunate and artemether. Because it is bom cheap and effective, it is beginning to be included in treatment schedules. However, it is not yet licensed for use in Australia, North America or Europe. Its main value is in the treatment of multi-drug resistant falciparum malaria. As the possibility of quinine resistance looms, artemisinin and its derivatives may soon become the drugs of choice for CM treatment.
  • Comparative analysis can be used for the diagnosis, monitoring, or prognosis of a malaria-related complication (including, for example, CM). Comparative analysis requires comparing one sample to another sample or reference point For example, comparisons can be between samples of the same subject, between samples of different subjects, between samples over time from the same subject, between samples over time from different subjects, between a sample and a reference point, etc.
  • a reference point generally refers to a value that is obtained from a sample or samples that have been used to arrive at a value that is indicative of a diagnostic, monitoring, or prognostic value.
  • a value that is indicative of diagnosing CM can be determined by obtaining a sample from a subject known to have CM and assessing the amount of the SUR1/TRPM4 channel, either qualitatively, quantitatively, or semi-quantitatively.
  • a reference point or sample can be used to indicate the absence of CM in a subject, and an amount of SUR1/TRPM4 in a test sample that is greater than the amount in the reference sample can be indicative of CM in the subject.
  • General aspects of the invention related to the diagnosis, monitoring, or prognosis of CM comprises certain steps.
  • steps include, for example, providing or obtaining a test sample; processing the same, if necessary, in preparation for deterrnining the quantity of the SUR1/TRPM4 channel; detecting an amount of the SUR1/TRPM4 channel either qualitatively, quantitatively, or semi-quantitatively; comparing the amount of the SUR1/TRPM4 channel in the test sample to a reference sample, and determining the diagnosis, monitoring, or prognosis of CM based on this comparison.
  • methods of diagnosing a malaria-related complication in a subject comprise (a) providing a test sample from a subject comprising an amount of SUR1/TRPM4; (b) detecting an amount of SUR1/TRPM4 in the test sample; and (c) comparing the amount of SUR1/TRPM4 in the test sample to an amount of SUR1/TRPM4 in a reference sample, wherein if there is a difference or a similarity in the amount of SUR1/TRPM4 in the test sample as compared with the amount of SUR1/TRPM4 in the reference sample, then a malaria-related complication is present in the subject.
  • the reference sample is from a subject that does not have cerebral malaria, and the amount of SUR1/TRPM4 in the test sample is greater than the amount of SUR1/TRPM4 in the reference sample, indicating the presence of cerebral malaria.
  • the reference sample is from a subject that has cerebral malaria
  • the amount of SUR1/TRPM4 in the test sample is similar to the amount of SUR1/TRPM4 in the reference sample, indicating the presence of cerebral malaria.
  • Detecting "SUR1/TRPM4" includes detecting SUR1, TRPM4 or both, and includes detection of nucleic acid or protein of SUR1/TRPM4, either directly or indirectly.
  • the SUR1/TRPM4 channel is not expressed unless a subject has malaria, in which case a malaria-related complication (including, for example, CM) may be or will be present
  • an increase in the quantity of the SUR1/TRPM4 channel when compared to the SUR1/TRPM4 channel in a reference sample indicates a diagnosis of a CM, a progression of CM, or an increased probability of suffering from CM.
  • an increase in the quantity of the SUR1/TRPM4 channel can be an increase from no detection (e.g., the SUR1/TRPM4 channel is not present, present in negligible quantities, or present in quantities that are not detectable) to detection of any measureable amount
  • an increase can be about 1-fold, 2-fold, 3-fold, 4-fold, 5- fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 11-fold, 12-fold, 13-fold, 14-fold, 15- fold, 16-fold, 17-fold, 18-fold, 19-fold, 20-fold, 21-fold, 22-fold, 23-fold, 24-fold, 25-fold, 26-fold, 27-fold, 28-fold, 29-fold, 30-fold, 31 -
  • an antagonist of the invention can be used for the diagnosis, monitoring, or prognosis of CM.
  • detecting expression of any portion of the SUR1/TRPM4 channel for example, expression of the regulatory subunit (including, for example, SUR1) and/or expression of the pore-forming subunit (including, for example, TRPM4), is used for the diagnosis, monitoring, or prognosis of CM. It is understood by the present invention that the up-regulation or increased expression of SUR1, TRPM4, or both, coincides with the up-regulation or increased expression of the SUR1/TRPM4 channel, which correlates to an increase in injury or likelihood of injury due to CM.
  • a conjugate of an antagonist of the invention can be used wherein the conjugate or channel itself is detected.
  • conjugates are those conjugates in which a molecule
  • Detectable labels are compounds and/or elements that can be detected due to their specific structural properties, functional properties, and/or chemical characteristics (by, for example, the use of an antibody specific for the detectable label).
  • Diagnostic agents generally fall within two classes, those for use in in vitro diagnostics, such as in a variety of immunoassays, and/or those for use in vivo diagnostic protocols, generally known as, for example, "molecule-directed imaging.”
  • imaging agents are known in the art, as are methods for their attachment to molecules, for example, antibodies (see, for e.g., U.S. Pat. Nos. 5,021,236; 4,938,948; and 4,472,509, each incorporated herein by reference).
  • the imaging moieties used can be, for example, paramagnetic ions; radioactive isotopes; fluorochromes; NMR-detectable substances; X-ray imaging.
  • Paramagnetic ions include, for example, ions such as chromium (III), manganese (II), iron (II ⁇ ), iron (II), cobalt (II), nickel (II), copper (II), neodymium (III), samarium ( ⁇ II), ytterbium (III), gadolinium (III), vanadium (II), terbium (ID), dysprosium (III), holmium (II ⁇ ) and/or erbium (III), with gadolinium being particularly preferred.
  • Ions useful in other contexts, such as X-ray imaging include but are not limited to lanthanum (III), gold (III), lead (II), and especially bismuth (III).
  • Radioactive isotopes for therapeutic and/or diagnostic application include, for example, 211 astatine, 11 carbon, 14 carbon, 51 chromium, 36 chlorine, 57 cobalt, 57 cobalt, 67 copper, 152 Eu, "gallium, 3 hydrogen, 123 iodine, 125 iodine, 131iodine, 111 indium, 59 iron, 32 phosphorus, 186 rhenium, 188 rhenium, 75 selenium, 35 sulphur, 99 technicium and/or 90 yttrium.
  • 125 I is often being preferred for use in certain embodiments, and 99 mtechnicium and/or 111 indium are also often preferred due to their low energy and suitability for long range detection.
  • fluorescent labels contemplated for use as conjugates include, for example, Alexa 350, Alexa 430, AMCA, BODIPY 630/650, BODIPY 650/665, BODIPY-FL, BODIPY-R6G, BODIPY-TMR, BODIPY-TRX, Cascade Blue, Cy3, Cy5,6-FAM, Fluorescein Isothiocyanate, HEX, 6-JOE, Oregon Green 488, Oregon Green 500, Oregon Green 514, Pacific Blue, REG, Rhodamine Green, Rhodamine Red, Renographin, ROX, TAMRA, TET, Tetramethylrhodamine, and/or Texas Red.
  • conjugate contemplated in the present invention are those intended primarily for use in vitro, where the molecule is linked to a secondary binding ligand and/or to an enzyme (an enzyme tag) that will generate a colored product upon contact with a chromogenic substrate.
  • suitable enzymes include urease, alkaline phosphatase, (horseradish) hydrogen peroxidase or glucose oxidase.
  • Preferred secondary binding ligands are biotin and/or avidin and streptavidin compounds. The use of such labels is well known to those of skill in the art and are described, for example, in U.S. Pat. Nos. 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149 and 4,366,241; each incorporated herein by reference.
  • Immunoassays in their most simple and direct sense, are binding assays. Certain preferred immunoassays are the various types of radioimmunoassays (RIA) and immunobead capture assay. Immunohistochemical detection using tissue sections also is particularly useful. However, it will be readily appreciated that detection is not limited to such techniques, and Western blotting, dot blotting, FACS analyses, and the like also may be used in connection with the present invention.
  • Immunologically-based detection methods for use in conjunction with Western blotting include enzymatically-, radiolabel-, or fluorescently-tagged secondary molecules/antibodies against regulatory subunit of the SUR1/TRPM4 channel, SUR1, and/or expression of the pore-forming subunit, TRPM4, is considered to be of particular use in this regard.
  • U.S. Patents concerning the use of such labels include U.S. Pat Nos. 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149 and 4,366,241, each incorporated herein by reference.
  • a secondary binding ligand such as a second antibody or a biotin/avidin ligand binding arrangement, as is known in the art.
  • PET scans involve the acquisition of physiologic images based on the detection of radiation from the emission of positrons.
  • Positrons are tiny particles emitted from a radioactive substance administered to the subject
  • an antagonist of the invention is enzymatically-, radiolabel-, or fluorescently-tagged, as described above and used to diagnosis, monitor, and/or prognosis of CM.
  • the method may comprise certain steps.
  • a biological sample is provided or obtained from a subject.
  • the biological sample may be tissue or fluid, and includes material that is physically isolated from the subject and can also include material that has not been physically isolated from a subject, for example, where expression levels are detected using in vivo techniques.
  • the biological sample includes cells from cerebrospinal fluid or cells from the brain and/or cerebral endothelial cells or microvessels and/or gliotic capsule of a CM patient or a patient suspected of having CM.
  • the nucleic acids used are isolated from cells contained in the biological sample, according to standard methodologies (e.g., Sambrook et al., Molecular Cloning: a laboratory manual. 2nd ed. N.Y., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, 1989).
  • the nucleic acid may be fractionated or whole cell RNA. It can be desirable to convert the RNA to a complementary DNA (cDNA).
  • cDNA complementary DNA
  • the RNA is whole cell RNA; in another, it is poly-A RNA.
  • the nucleic acid is amplified before detection.
  • the specific nucleic acid of interest is identified in the sample directly using amplification or with a second, known nucleic acid following amplification.
  • the identified product is detected.
  • the detection may be performed by visual means (e.g., ethidium bromide staining of a gel).
  • the detection may involve indirect identification of the product via chemiluminescence, radioactive scintigraphy of radiolabel or fluorescent label or even via a system using electrical or thermal impulse signals (Affymax Technology; Bellus, 1994).
  • chip-based DNA technologies such as those described by Hacia et al., Nature Genet 14 : 441-447 (1996) and Shoemaker et al., Nature Genetics 14: 450-456 (1996) can be used for diagnosis, monitoring, or prognosis. Briefly, these techniques involve quantitative methods for analyzing large numbers of genes rapidly and accurately. By tagging genes with oligonucleotides or using fixed probe arrays, one can employ chip technology to segregate target molecules as high density arrays and screen these molecules on the basis of hybridization. See also Pease et al., Proc. Natl. Acad Sci. USA 91: 5022- 5026 (1994); Fodor et al., Science 251: 767-773 (1991).
  • kits for treating CM comprising an inhibitor of SUR1/TRPM4 channel, including an inhibitor of SUR1 and/or TRPM4.
  • the channel inhibitor is, for example, a SUR1 inhibitor, a TRPM4 inhibitor, or a mixture or combination thereof.
  • a kit of the invention can also include an additional reagent. Reagents that are suited for obtaining blood or plasma or serum from an individual may be included in a kit of the invention, such as a syringe, collection vial, needle, and so forth.
  • kits may comprise a suitably aliquoted composition and/or additional agent compositions of the present invention, whether labeled or unlabeled, as may be used to prepare a standard curve for a detection assay.
  • the components of the kit may be packaged in combination or alone in the same or in separate containers, depending on, for example, cross-reactivity or stability, and can also be supplied in solid, liquid, lyophilized, or other applicable form.
  • the container means of the kits will generally include, for example, at least one vial, test tube, flask, bottle, syringe or other container means, into which a component may be placed, and preferably, suitably aliquoted.
  • kits of the present invention can contain a second, third or other additional container into which the additional component may be contained.
  • additional component may be contained in various combinations.
  • kits of the present invention also will typically include a means for containing the composition, additional agent, and any other reagent containers in close confinement for commercial sale.
  • Such containers may include, for example, injection or blow molded plastic or glass containers into which the desired vials are retained.
  • the liquid solution is an aqueous solution, with a sterile aqueous solution being particularly preferred.
  • the compositions may also be formulated into a syringeable composition.
  • the container means may itself be a syringe, pipette, and/or other such like apparatus, from which the formulation may be applied to an infected area of the body, injected into an animal, and/or even applied to and/or mixed with the other components of the kit.
  • the components of the kit may be provided as dried powder(s).
  • the powder can be reconstituted by the addition of a suitable solvent
  • the solvent may also be provided in another container means.
  • the container means will generally include at least one vial, test tube, flask, bottle, syringe and/or other container means, into which the composition is placed, preferably, suitably allocated.
  • the kits may also comprise a second container means for containing a sterile, pharmaceutically acceptable buffer and/or other diluent.
  • kits of the present invention will also typically include a means for containing the vials in close confinement for commercial sale, such as, e.g., injection and/or blow-molded plastic containers into which the desired vials are retained.
  • a means for containing the vials in close confinement for commercial sale such as, e.g., injection and/or blow-molded plastic containers into which the desired vials are retained.
  • the kits of the invention may also comprise, and/or be packaged with, an instrument for assisting with the mjection/adniinistration and/or placement of the composition within the body of an animal.
  • an instrument may be a syringe, pipette, forceps, and/or any such medically approved delivery vehicle.
  • Example 1 Expression of SUR1 in Brain Tissues from Mice Infected with P. Berghei ANKA.
  • mice were infected with P. Berghei ANKA parasites for 6-10 days. Brain tissue was taken from infected mice and analyzed for the presence of SUR1 , which is a marker for the SUR1/TRPM4 channel. Samples were taken from a mouse with systemic malaria that did not exhibit cerebral involvement and from another mouse with systemic malaria that was moribound, a cardinal manifestation of CM. There was no detection of SUR1 in the mouse with systemic malaria that did not exhibit cerebral involvement, but there was detection of SUR1 in the mouse with systemic malaria that was moribound (Figure 1).
  • SUR1 the regulatory subunit of the SUR1/TRPM4 channel
  • CM the regulatory subunit of the SUR1/TRPM4 channel
  • Example 2 Expression of the SUR1/TRPM4 Channel in Human Malaria Infected Brain.
  • the presence of the SUR1/TRPM4 channel in CM patients supports detecting the SUR1/TRPM4 channel (e.g., by detecting SUR1 or TRPM4 subunits) for the diagnosis, monitoring, and prognosis of CM.
  • Example 3 Glibenclamide Improves CM-related Survival and Neurological Function.
  • a rat model of cerebral malaria based on Plasmodium berghei in which neurological symptoms develop that are associated with sequestration of parasitized erythrocytes and leukocytes and with clogging and rupture of vessels in both cerebral and cerebellar regions is used (Hearn et al., Infect. Immun. 68, 5364 (2000); Pedroni et al., Exp. Parasitol. 113, 193 (2006)).
  • Another animal mat can be used is Plasmodium coatneyi infected rhesus monkeys (Aikawa et al., Am. J. Trap. Med Hyg., 46(4), 1992, pp. 391-397).
  • the brain tissues were immunolabeled from these patients for Surl using a custom anti-Surl antibody validated for human tissues (Simard et al., Sci. Transl. Med. 2, 28ra29 (2010)).
  • Fig. 2B Abundant expression of Surl in tissues from all of these patients was observed (Fig. 2B), but not in tissues from control patients.
  • Surl was upregulated in microvascular endothelium (Fig. 2D, arrowheads), which is responsible for edema formation and secondary hemorrhage.
  • Surl was upregulated in neurons (Fig. 2D, asterisks) and was localized to the parasites themselves (Fig. 2D, arrows).
  • Sur1 blocker glibenclamide may be an important new treatment to ameliorate the complications of cerebral malaria, including cerebral edema, secondary hemorrhage and death.

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Abstract

L'infection de type malaria reste la maladie parasitaire la plus répandue au monde qui affecte le système nerveux central. Des centaines de millions d'individus sont infectés chaque année, occasionnant plus de 1 million de décès, ce qui constitue un problème médical, économique et émotionnel majeur au sein des pays en voie de développement. Une conséquence significative de l'infection de type malaria est la malaria cérébrale (MC). Il est clair qu'il existe un besoin médical non satisfait de traiter la MC ainsi que d'autres conséquences de l'infection de type malaria. À cette fin, la présente invention divulgue ici des nouveaux procédés et des nouvelles compositions pour traiter une complication associée à la malaria, y compris des procédés et des compositions pour traiter la MC.
PCT/US2010/060565 2009-12-16 2010-12-15 Procédés et compositions pour traiter une complication associée à la malaria Ceased WO2011084514A2 (fr)

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