WO2007114948A2 - Méthodes et compositions pour inhiber la mort cellulaire - Google Patents

Méthodes et compositions pour inhiber la mort cellulaire Download PDF

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Publication number
WO2007114948A2
WO2007114948A2 PCT/US2007/008507 US2007008507W WO2007114948A2 WO 2007114948 A2 WO2007114948 A2 WO 2007114948A2 US 2007008507 W US2007008507 W US 2007008507W WO 2007114948 A2 WO2007114948 A2 WO 2007114948A2
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release
cytochrome
inhibiting compound
group
cell death
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WO2007114948A3 (fr
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Robert M. Friedlander
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Brigham and Womens Hospital Inc
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Brigham and Womens Hospital Inc
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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
    • A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
    • A61K31/403—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil condensed with carbocyclic rings, e.g. carbazole
    • A61K31/404—Indoles, e.g. pindolol
    • A61K31/405—Indole-alkanecarboxylic acids; Derivatives thereof, e.g. tryptophan, indomethacin

Definitions

  • the invention relates to methods and products for modulating mitochondrial cytochrome c release and for inhibiting cell death.
  • the invention is useful for preventing and treating cell death-associated disorders such as degenerative diseases and disorders, including, but not limited to Alzheimer's disease, Parkinson's disease; Huntington's disease; Amyotrophic Lateral Sclerosis (ALS), epilepsy; stroke, head trauma; spinal cord injury; perinatal hypoxic injury; retinal degeneration; cerebral or other end-organ ischemia inducing cell death associated with a neurosurgical, vascular, cardiac, or transplantation surgery; myocardial infarction; or diabetes, etc.
  • degenerative diseases and disorders including, but not limited to Alzheimer's disease, Parkinson's disease; Huntington's disease; Amyotrophic Lateral Sclerosis (ALS), epilepsy; stroke, head trauma; spinal cord injury; perinatal hypoxic injury; retinal degeneration; cerebral or other end-organ ischemia inducing cell death associated with a neurosurgical, vascular, cardiac, or transplantation surgery; myocardi
  • compositions and methods of the invention can also be used to prevent and/or treat cell death associated with carotid endarterectomy; carotid and cardiac stents; and abdominal aortic aneurism (AAA) repair.
  • the invention also relates in part to assays that are useful for identifying and testing candidate compounds for modulating mitochondrial cytochrome c release.
  • Cytochrome c release from mitochondria is critical and common to neuronal death following various insults to the CNS during acute and chronic neurodegeneration (Friedlander, 2003; Hengartner, 2000; Rigamonti et al., 2001 ; Wang et al., 2003; Zhang et al., 2003a; Zhu et al., 2002).
  • an oligomeric complex called the apoptosome is assembled consisting of cytochrome c, pro-caspase-9 and Apaf- 1. Assembly of the apoptosome results in caspase-9 activation, which thereafter activates caspase-3 leading to cell death (Li et al., 1997; Zou et al., 1997).
  • Drugs capable of inhibiting mitochondrial release of cytochrome c would be attractive targets for treatment of neurologic diseases featuring caspase-mediated cell death.
  • the Food and Drug Administration (FDA) approved drugs have been identified that have unexpected properties of being able to modulate mitochondrial cytochrome c release.
  • the invention relates in part to the use of these newly identified compounds in methods and compositions to prevent and/or treat cell death in neurodegenerative diseases and disorders.
  • fendiline hydrochoride calcimycin, dipyrone, ritanserin, azathioprine, bepridil hydrohloride, parthenolide, probucol, methazolamide, hydroxy-progesterone caproate, doxycycline hydrochloride, melatonin, minocycline hydrochloride, mephenytoin, and N-acetyl-DL-tryptophan as compounds that modulate mitochondrial cytochrome c release.
  • Fendiline hydrochoride, calcimycin, dipyrone, ritanserin, azathioprine, bepridil hydrohloride, parthenolide, probucol, methazolamide, hydroxy-progesterone caproate, doxycycline hydrochloride, melatonin, minocycline hydrochloride, mephenytoin, and N-acetyl-DL-tryptophan have not been used in methods to modulate cytochrome c release and the invention includes the use of these compounds to inhibit cytochrome c release and in the treatment of cell death-associated disorders and diseases.
  • the invention includes, in part, methods for treating disorders resulting from aberrant cytochrome c release and compositions for treating such disorders.
  • methods for assaying candidate compounds for the ability to inhibit cytochrome c release we have identified methods of assaying candidate compounds for the ability to inhibit cytochrome c release.
  • methods for preventing or treating a cell-death associated-disease or disorder in a subject include administering to a subject in need of such treatment an effective amount of a Group A and/or a Group B cytochrome c release-inhibiting compound to treat the cell death-associated disease or disorder, wherein the subject is otherwise free of indications for treatment with the Group A or Group B cytochrome c release-inhibiting compound that is administered.
  • a group A cytochrome c release-inhibiting compound is administered.
  • the Group A cytochrome c release-inhibiting compound is calcimycin, or an analog, derivative, or variant thereof that inhibits cytochrome c release.
  • the Group A cytochrome c release-inhibiting compound is dipyrone, or an analog, derivative, or variant thereof that inhibits cytochrome c release. In some embodiments, the group A cytochrome c release-inhibiting compound is parthenolide, or an analog, derivative, or variant thereof that inhibits cytochrome c release. In some embodiments, the Group A cytochrome c release-inhibiting compound is hydroxyprogesterone caproate, or an analog, derivative, or variant thereof that inhibits cytochrome c release. In certain embodiments, the Group A cytochrome c release-inhibiting compound is mephenytoin, or an analog, derivative, or variant thereof that inhibits cytochrome c release.
  • the Group A cytochrome c release-inhibiting compound is N-acetyl-DL-tryptophan, or an analog, derivative, or variant thereof that inhibits cytochrome c release.
  • the cell death-associated disease or disorder is Alzheimer's disease, Parkinson's disease; Huntington's disease; Amyotrophic lateral sclerosis (ALS), epilepsy; stroke, head trauma; spinal cord injury; perinatal hypoxic injury; retinal degeneration; cerebral or other end-organ ischemia inducing cell death associated with a neurosurgical, vascular, cardiac, or transplantation surgery; myocardial infarction, or diabetes.
  • the subject is human.
  • the cytochrome c release-inhibiting compound is linked to a targeting molecule.
  • the targeting molecule's target is a neuronal cell.
  • the cytochrome c release-inhibiting compound is administered prophylactically to a subject at risk of having a cell death-associated disease or disorder.
  • the cytochrome c release-inhibiting compound is administered in combination with an additional drug for treating a cell death-associated disease or disorder.
  • the. methods also include administering a Group B cytochrome c release-inhibiting compound.
  • the Group B cytochrome c release-inhibiting compound is fendiline hydrochloride, ritanserin, azathioprine, bepridil hydrochloride, probucol, methoazolamide, doxycycline hydrochloride, melatonin, or minocycline hydrochloride, or an analog, derivative, or variant of fendiline hydrochloride, ritanserin, azathioprine, bepridil hydrochloride, probucol, methoazolamide, doxycycline hydrochloride, melatonin, or minocycline hydrochloride that inhibits cytochrome c release.
  • methods of evaluating the effect of candidate pharmacological agents on cytochrome c release include contacting a cell-free sample comprising mitochondria with a candidate pharmacological agent; determining the effect of the candidate pharmacological agent on the level of cytochrome c release in the cell-free sample relative to the level of cytochrome c release control cell-free sample not contacted with the candidate pharmacological agent, wherein a relative decrease in the level of cytochrome c release in the cell-free sample as compared with the level in the control cell-free sample indicates the inhibition of cytochrome c release by the candidate pharmacological agent.
  • the cell-free sample is contacted with calcium in an amount effective to include cytochrome c release in a control sample.
  • the amount of cytochrome c release is determined using an ELISA assay.
  • a relative decrease in the level of cytochrome c release in the cell-free sample contacted with the candidate pharmacological agent compared to the level of cytochrome c release in the cell-free sample not contacted with the candidate pharmacological agent indicates the candidate pharmacological agent is a cytochrome c release-inhibiting compound.
  • kits for treating a subject using any of the aforementioned aspects of the invention include a package housing a first container containing at least one dose of a Group A or a Group B cytochrome c release-inhibiting compound, and instructions for using the cytochrome c release-inhibiting compound in the prevention and/or treatment of an cell death associated disease or disorder.
  • the container contains at least one dose of a Group A cytochrome c release-inhibiting compound.
  • the Group A cytochrome c release-inhibiting compound is calcimycin, or an analog, derivative, or variant thereof that inhibits cytochrome c release.
  • the Group A cytochrome c release-inhibiting compound is dipyrone, or an analog, derivative, or variant thereof that inhibits cytochrome c release. In some embodiments, the Group A cytochrome c release-inhibiting compound is parthenolide, or an analog, derivative, or variant thereof that inhibits cytochrome c release. In certain embodiments, the Group A cytochrome c release-inhibiting compound is hydroxyprogesterone caproate, or an analog, derivative, or variant thereof that inhibits cytochrome c release. In some embodiments, the Group A cytochrome c release-inhibiting compound is mephenytoin, or an analog, derivative, or variant thereof that inhibits cytochrome c release.
  • the Group A cytochrome c release-inhibiting compound is N-acetyl-DL-tryptophan, or an analog, derivative, or variant thereof that inhibits cytochrome c release.
  • the cell death-associated disease or disorder is Alzheimer's disease, Parkinson's disease; Huntington's disease; ALS, epilepsy; stroke, head trauma; spinal cord injury; perinatal hypoxic injury, retinal degeneration; cerebral or other end-organ ischemia inducing cell death associated with a neurosurgical, vascular, cardiac, or transplantation surgery; myocardial infarction; or diabetes.
  • the kit also includes a container containing at least one dose of a Group B cytochrome c release-inhibiting compound, wherein the compound is fendiline hydrochloride, ritanserin, azathioprine, bepridil hydrochloride, probucol, methoazolamide, doxycycline hydrochloride, melatonin, or minocycline hydrochloride, or an analog, derivative, or variant of fendiline hydrochloride, ritanserin, azathioprine, bepridil hydrochloride, probucol, methoazolamide, doxycycline hydrochloride, melatonin, or minocycline hydrochloride that inhibits cytochrome c release and instructions for using the compound for prevention and/or treatment of a cell death associated disease or disorder.
  • a Group B cytochrome c release-inhibiting compound wherein the compound is fendiline hydrochloride, ritanserin,
  • the cytochrome c release-inhibiting compound is formulated for delivery to neuronal cells. In some embodiments, the cytochrome c release-inhibiting compound is formulated for sustained release. In certain embodiments, the cytochrome c release-inhibiting compound is lyophilized.
  • compositions include a Group A cytochrome c release-inhibiting compound and a Group B cytochrome c release-inhibiting compound.
  • the Group A cytochrome c release-inhibiting compound is calcimycin, dipyrone, parthenolide, hydroxyprogesterone caproate, mephenytoin, or N-acetyl-DL-tryptophan, or an analog, derivative, or variant of calcimycin, dipyrone, parthenolide, hydroxyprogesterone caproate, mephenytoin, or N-acetyl-DL-tryptophan that inhibits cytochrome c release.
  • the Group B cytochrome c release-inhibiting compound is fendiline hydrochloride, ritanserin, azathioprine, bepridil hydrochloride, probucol, methoazolamide, doxycycline hydrochloride, melatonin, or minocycline hydrochloride, or an analog, derivative, or variant of fendiline hydrochloride, ritanserin, azathioprine, bepridil hydrochloride, probucol, methoazolamide, doxycycline hydrochloride, melatonin, or minocycline hydrochloride that inhibits cytochrome c release.
  • the cytochrome c release-inhibiting compound is linked to a targeting molecule.
  • the targeting molecule's target is a neuronal cell.
  • the cytochrome c release-inhibiting compound is formulated for implantation, mucosal administration, injection, inhalation, or oral administration.
  • therapeutic methods include administering a Group A cytochrome c release-inhibiting compound to a subject based on a diagnosis of an cell death and/or cytochrome c release-associated disease in the subject, in an amount effective to treat the disease.
  • the Group A cytochrome c release-inhibiting compound is calcimycin, or an analog, derivative, or variant thereof.
  • the Group A cytochrome c release-inhibiting compound is dipyrone, or an analog, derivative, or variant thereof.
  • the Group A cytochrome c release-inhibiting compound is parthenolide, or an analog, derivative, or variant thereof.
  • the Group A cytochrome c release-inhibiting compound is hydroxyprogesterone caproate, or an analog, derivative, or variant thereof. In some embodiments, the Group A cytochrome c release-inhibiting compound is mephenytoin, or an analog, derivative, or variant thereof. In some embodiments, the cytochrome c release-inhibiting compound is N-acetyl-DL-tryptophan, or an analog, derivative, or variant thereof.
  • the cell death and/or cytochrome c release-associated disease, disorder, or procedure is Alzheimer's disease, Parkinson's disease; Huntington's disease; ALS, epilepsy; stroke, head trauma; spinal cord injury; perinatal hypoxic injury; retinal degeneration; cerebral or other end-organ ischemia inducing cell death associated with a neurosurgical, vascular, cardiac, or transplantation surgery; myocardial infarction; perioperative neuroprotection during carotid endarterectomy; carotid and cardiac stents; abdominal aortic aneurism (AAA) repair; or diabetes.
  • the subject is human.
  • the cytochrome c release- inhibiting compound is linked to a targeting molecule.
  • the targeting molecule's target is a neuronal cell.
  • the cytochrome c release-inhibiting compound is administered prophylactically to a subject at risk of having a cell death associated disease or disorder and/or a cytochrome c release-associated disease or disorder.
  • the mode of administration is selected from the group consisting of: implantation, mucosal administration, injection, inhalation, and oral administration.
  • the cytochrome c release-inhibiting compound is administered in combination with an additional drug for treating a cell death-associated disease or disorder and/or a cytochrome c release-associated disease or disorder.
  • aspects of the invention also include administering a cytochrome c release-inhibiting compound selected from the group consisting of Group B cytochrome c release-inhibiting compounds.
  • the Group B cytochrome c release-inhibiting compound is fendiline hydrochloride, ritanserin, azathioprine, bepridil hydrochloride, probucol, methoazolamide, doxycycline hydrochloride, melatonin, or minocycline hydrochloride, or an analog, derivative, or variant of fendiline hydrochloride, ritanserin, azathioprine, bepridil hydrochloride, probucol, methoazolamide, doxycycline hydrochloride, melatonin, or minocycline hydrochloride that inhibits cytochrome c release.
  • compositions in the preparation of a medicament, particularly a medicament for prevention and/or treatment of a cytochrome c release-associated disease or disorder and/or a cell death-associated disease or disorder.
  • Fig. 1 shows two graphs and a list of experimental results indicating that cytochrome c release is differentially affected by various compounds.
  • Fig. Ia shows overall results of how 80 compounds in kit 6 of the NINDS library were scored.
  • the green line marks the level of cytochrome release upon stimulation with Ca 2+ ions in the absence of additional pharmacologic agents. Compounds scored in the interval marked by the green line had no effect on cytochrome release; those scored to its left were inhibitory of the process, and those scored to its right are stimulatory. Additional controls were the levels of cytochrome c release when Ca 2+ was absent (blue line) and when Ca 2+ -stimulated release was countered by treatment with cyclosporin A (red line).
  • Fig. Ib is a graph showing colored curves that represent distributions of drug potencies for the 13 kits with 80 compounds in each. In each kit, levels of cytochrome c release were normalized relative to the average of those 80 values. The black line is the sum of these 13 distributions. The dotted line is 1.5 standard deviations less than the mean in the grand distribution.
  • the X-axis was divided into intervals, each spanning l/20th of the unit distance from zero to the normalized mean.
  • the Y-axis reports the number of measurements (out of the set of 80) that fall into the interval in question.
  • Fig. Ic shows the ranking by effectiveness at inhibiting cytochrome c release from purified mitochondria; of the 21 compounds that were selected. All are in clinical usage, each in the indicated capacity. Whenever known, the molecular target was noted.
  • Fig. 2. shows graphs and tables of results of screens of neuroprotection ability of 21 compounds in cultured cells. Mutant-htt ST14A striatal cells were shifted to non-permissive temperature to 37°C in serum-deprived medium (SDM) with or without different test drugs for 18 h. Relative cell death was evaluated by the
  • Test drugs are divided into group I (Fig. 2a), II (Fig. 2b), and HI (Fig. 2c) according to their ability to inhibit cell death and IC 50 range.
  • the calculated IC 50 , maximum protection, and/or toxicity afforded by each drug in the different groups are listed in the lower panel, respectively.
  • Fig. 3 shows graphs and digitized microscopic images of neuronal cells demonstrating that Methazolamide and melatonin inhibit neuronal cell death by preventing the dissipation of ⁇ m; methazolamide and melatonin do not inhibit mPT.
  • Fig. 3a shows results of PCNs that were treated with 1 raM H 2 O 2 or 500 ⁇ M NMDA for 18 h or kept 18 h after 3 h of OGD treatment in the presence or absence of indicated concentration of methazolamide or melatonin or minocycline or 100 ⁇ M test drug in OGD treatment. Cell death was evaluated by the LDH assay (*, P ⁇ 0.05; **, P ⁇ 0.001).
  • Figs. 3b and 3c show results of PCNs treated with OGD (Fig. 3b) or striatal cells were shifted at the non-permissive temperature of 37°C in SDM (Fig. 3c) with or without methazolamide or melatonin or gossypol-acetic acid complex for 5 h and 18 h. Living cells were stained directly with 2 ⁇ M RH 123. Arrows show the dissipation of ⁇ m (scale bar, 5 ⁇ m). Figs.
  • Fig. 4 shows brain section digitized images, graphs, and Western blots indicating results of the characterization of neuroprotection of methazolamide and melatonin in cerebral ischemia in vivo.
  • Lesional sizes Figs. 4a and 4b
  • neuro-scores Fig. 4c
  • a vehicle stroke group or groups of mice treated with methazolamide (20 mg/kg weight) and melatonin (10 mg/kg weight) at indicated times pre- (1 h) or post- (30 min) MCAO (n 6 or 7 mice per condition, *, P ⁇ 0.05, **, P ⁇ 0.001).
  • Fig. 4a is the staining examples
  • Fig. 4d cytosolic fraction lysates
  • Fig. 4e brain lysates
  • the protein samples were separated by SDS/PAGE and probed respectively with antibodies to cytochrome c or to caspase-3.
  • the example of Western blotting was selected as no cerebral ischemia, cerebral ischemia, cerebral ischemia with the addition of methazolamide (post-treatment) or melatonin (pre-treatment); the same blot was reprobed with /3-actin antibody (Figs. 4d and 4e).
  • Empty bars indicate samples without cerebral ischemia and test drugs.
  • Black solid bars indicate samples with cerebral ischemia.
  • Blue or green bars indicate samples with cerebral ischemia plus test drug by pre-treatment or post-treatment, respectively.
  • Fig. 5 shows drawings of the chemical structure of 21 "hit” compounds are shown as groups I, ⁇ , and m.
  • Fig. 6 shows micrographic images of cells, western blots and graphs indicting that methazolamide and melatonin inhibit the release of mitochondrial apoptogenic factors and forestall the activation of caspase-9 and -3.
  • Fig. 6 A shows cell death is induced in mutant-htt striatal cells by shifting them to the nonpermissive temperature of 37°C in SDM. Test cell cultures contain 100 ⁇ M methazolamide or melatonin, whereas controls are devoid of these drugs. After 5 hours, the cells are stained with Mitotracker and then fixed and stained with antibodies to cytochrome c. Mitochondrial cytochrome c is demonstrated as having a punctate pattern that colocalizes with Mitotracker.
  • Fig. 6b shows results of mutant-htt striatal cells that were treated with 100 ⁇ M methazolamide or melatonin (or processed in the absence of a test drug) for 18 hours. Subsequently, they were extracted and the resulting lysate were separated into cytosolic components. The samples, each of which contains 50 ⁇ g of protein, were analyzed by Western blot using antibodies to cytochrome c, Smac, caspase-9, or caspase-3.
  • j3-actin was used as a loading control.
  • This blot is representative of three independent experiments.
  • Figs. 6c and d show results of mutant-htt striatal cells that were treated with 100 ⁇ M methazolamide or melatonin for 5 and 18 hours and compared to untreated controls. Cells were lyzed and their complement of caspase-9 and —3 activities were evaluated by a fluorogenic assay. The results are the mean ⁇ SD of three independent experiments (* indicates that/? ⁇ 0.05, ** that it/? ⁇ * 0.001).
  • Experimental controls include cells treated with neither a cell death stimulus nor a test drugs (white bars). Others that receive a death stimulus but are again not treated with a test drug (black bars).
  • Fig. 7 shows micrographic images of cells and graphs indicting that methazolamide and melatonin slow the dissipation of ⁇ m , but do not inhibit mPT.
  • the culture medium was supplemented with methazolamide or melatonin (test drugs) on the one hand, or with gossypol-acetic acid complex or no drug whatsoever (negative controls) on the other.
  • the living cells were then stained with 2 ⁇ M Rh 123 in order to determine the electrostatic charge of the mitochondria. (Fig. 7c,d)
  • methazolamide nor melatonin prevented the induction of the mPT as judged by an unchanging degree of mitochondrial swelling.
  • Liver mitochondria (0.25 mg/ml) were energized with 5 mM glutamate/malate and incubated with 1 , 5, 10 and 20 ⁇ M of methazolamide (Fig. 7E) or melatonin (Fig. 7F) in buffer containing 250 mM Sucrose, 10 mM HEPES, 2 mM KH 2 PO 4 .
  • Mitochondria were challenged with bolus additions of 5 ⁇ M Ca J+ every 2 min until release of sequestered Ca 1+ occurred.
  • Alameticin 100 ⁇ g was added in the end of each sample.
  • Upper left panel of Fig. 7e and f is ⁇ m
  • upper right is in the buffer
  • lower left NADH level
  • lower right is swelling. See methods for additional information.
  • FIG. 8a Brains were quickly removed, cut into coronal sections, and stained with 2,3,5-triphenyltetrazolium chloride (Fig. 8a).
  • lysates of brain tissue were resolved into cytosolic fractions for analysis by western blotting (Fig. 8 d,e).
  • the protein samples were resolved on SDS/PAGE gels, transferred to nitrocellulose, probed with antibodies to cytochrome c or caspase-3, and reprobed with anti-j3-actin.
  • Western blots revealed changes in the cellular localization and degree of maturation of the respective target proteins. (Fig. 8 d,e).
  • Densitometric scans of these gels allowed the signals from cytoplasmic cytochrome c and activated caspase-3 to be compared to that from jS-actin.
  • Each test group consisted of 3-5 mice. Again, * indicates that/? ⁇ 0.05 and ** that/? ⁇ 0.001.
  • White bars correspond to brain samples from animals that neither underwent MCAO nor received any test drug.
  • Black bars correspond to samples from saline-injected animals that did undergo MCAO.
  • Blue and green bars correspond to samples from test animals, i.e., one that were both treated with an experimental drug and underwent MCAO.
  • Fig. 9 shows histograms and a graph demonstration the neuroprotection by dipyrone of cells subjected to OGD and of animals that underwent MCAO.
  • PCNs were incubated for 18 hours with the indicated concentrations of dipyrone starting an hour before a 3 hour of OGD. Control cells were treated identically but in the absence of dipyrone. Cell death was evaluated by the LDH assay (Fig. 9 a). A semi-logarithmic plot of the extent of cell death vs. drug concentration was fitted to a sigmoidal curve. The ICso and maximum protection were then calculated using a computer algorithm (Fig. 9b). White bars correspond to untreated cells that did not undergo OGD.
  • Black bars correspond to untreated cells that underwent 3 hours of OGD.
  • Grey bars corresponding dipyrone-treated cell that underwent 3 hours of OGD.
  • Fig. 9c,d Lesion size (Fig. 9c) and neuro-scores (Fig. 9d) were assessed 24 hours after cerebral ischemia.
  • Fig. 10 shows graphs, a histogram, and digitized images of brain sections demonstrating that methazolamide delays the onset of neurodegeneration in R6/2 mice.
  • FIG. 10 a, b Motor performance of R6/2 mice was evaluated by recording the time (up to 7 min) that they remained on a rotarod turning at 15 rpm (Fig. 10a) and 5 rpm (Fig. 10b). Mice were injected daily with 20 mg/kg or 40mg/kg methazolamide (or the saline vehicle) from 6 weeks of age. Measurements were made weekly beginning when the animals were 6 weeks old./? value is compared with saline-treated mice, for 20 mg/kg methazolamide,/?
  • Fig. 11 shows a table demonstrating results of Huntington's mice treated with melatonin at 30 mg/dosage versus saline-treated control mice.
  • Fig. 12 shows a table demonstrating results of Huntington's mice treated with melatonin at 10 mg/kg dosage versus saline-treated control mice.
  • Fig. 13 shows graphs demonstrating the results in mice treated with melatonin.
  • Fig. 13A shows results of mice on the rotarod at 15 rpm.
  • Fig. 13B shows results of mice on the rotarod at 5 rpm.
  • Fig. 13C shows body weight comparison between saline control animals and mice treated with melatonin.
  • compositions of the invention include compounds that inhibit the level of cytochrome c release and/or cell death in cells, tissues, and subjects.
  • cytochrome c release-inhibiting compounds means compounds that reduce the level of release of cytochrome c from mitochondria in cells, tissues, and subjects.
  • the methods of the invention involve the administration of cytochrome c release-inhibiting and therefore are useful to reduce or prevent cell death in various degenerative diseases and disorders.
  • cell death-associated disorder and “cytochrome c release-associated disorder” include, but are not limited to: Alzheimer's disease, Parkinson's disease; Huntington's disease; ALS, epilepsy; stroke, head trauma; spinal cord injury; perinatal hypoxic injury; retinal degeneration; cerebral or other end-organ ischemia inducing cell death associated with a neurosurgical, vascular, cardiac, or transplantation surgery; myocardial infarction; or diabetes. It will be understood that a cell death-associated disorder may result from a medical procedures and/or devices. The methods and compositions of the invention may also be used to prevent or cell death-associated disorders that occur as a result of medical procedures (e.g. surgery, stents, etc.).
  • medical procedures e.g. surgery, stents, etc.
  • Examples of types of procedures that may result in cell death treatable by the methods and compositions of the invention include, but are not limited to, end-organ ischemia associated with a neurosurgical, vascular, cardiac, or transplantation surgery, carotid endarterectomy; carotid and cardiac stents; and abdominal aortic aneurism (AAA) repair.
  • end-organ ischemia associated with a neurosurgical, vascular, cardiac, or transplantation surgery
  • carotid endarterectomy carotid and cardiac stents
  • abdominal aortic aneurism (AAA) repair abdominal aortic aneurism
  • compositions of the invention include compounds that inhibit the release of cytochrome c in cells and/or tissues, thereby reducing the cell and tissue damage and clinical manifestations of cytochrome c release-associated diseases and/or disorders.
  • the term "subject” means any mammal that may be in need of treatment with a cytochrome c release-inhibiting compound of the invention.
  • Subjects include but are not limited to: humans, non-human primates, cats, dogs, sheep, pigs, horses, cows, rodents such as mice, rats, etc.
  • Table 1 provides examples of cytochrome c release-inhibiting compounds of the invention.
  • the compounds calcimycin, dipyrone, parthenolide., hydroxyprogesterone caproate mephenytoin, and N-acetyl-DL-tryptophan, are known to act as agents in treatment of non-cytochrome c release associated diseases and/or disorders.
  • the foregoing compounds have never before been given to patients to reduce or inhibit cytochrome c release in cells and/or tissues, and/or to treat or prevent a cytochrome c release-associated disorder in subjects who are otherwise free of indications for their administration.
  • the calcimycin, dipyrone, parthenolide, hydroxyprogesterone caproate, mephenytoin, N-acetyl-DL-tryptophan, fendiline hydrochloride, ritanserin, azathioprine, bepridil hydrochloride, probucol, methoazolamide, doxycycline hydrochloride, melatonin, and minocycline hydrochloride compounds of the invention are administered to subjects that are free of indications for their previously determined use.
  • the term "previously determined use” of a compound means the use of the compound that was previously identified. Thus, the previously determined use is not the use of inhibiting cytochrome c release in cells and/or tissues.
  • the methods of the invention include administration of a cytochrome c release-inhibiting compound that preferentially targets neuronal cells and/or tissues or other specific tissues types.
  • the compounds can be specifically targeted to neuronal tissue (e.g. glial cells and/or neuronal cells) or other specific tissue types.
  • the targeting may be done using various delivery methods, including, but not limited to: administration to neuronal tissue or other specific target tissue, the addition of targeting molecules to direct the compounds of the invention to neuronal or other tissues (e.g. glial cells, neuronal cells, liver cells, heart cells, etc.). Additional methods to specifically target molecules and compositions of the invention to brain tissue and/or neuronal tissues or other tissue types are known to those of ordinary skill in the art.
  • the invention involves, in part, the administration of a compound that inhibits release of cytochrome c in cells, tissues, and/or subjects.
  • release means the release of cytochrome c from mitochondria. It is understood that the release of cytochrome c from mitochondria may occur following various insults to the CNS during acute and chronic neurodegeneration. The release of cytochrome c from mitochondria of other tissues (e.g. liver, cardiac, vascular, etc) also may result following injury or insult. Release of cytochrome c may result in assembly of the apoptosome, which results in caspase-9 activation, which thereafter activates caspase-3 leading to cell death. The release of cytochrome c from mitochondria is associated with cell death-associated diseases and disorders.
  • the term “inhibit” means to reduce the amount of cytochrome c release to a level or amount that is statistically significantly less than a control level of cytochrome c release.
  • the decrease in the level of cytochrome c release means the level of cytochrome c release is reduced from an initial level to a level significantly lower than the initial level, hi some cases this reduced level may be zero.
  • a control level of cytochrome c release is the level that represents the normal level of cytochrome c release in a cell, tissue, and/or subject.
  • a control level may be a level that is not associated with cell death.
  • a control level will be the level in a disorder-free cell, tissue, or subject, and maybe useful, for example, to monitor an increase in the level of cytochrome c release in a cell.
  • a control level of cytochrome c release will be the level in a cell, tissue, or subject with a neurological disorder, e.g. Alzheimer's disease, or ischemia, and may be useful, for example, to monitor a decrease in the level of cytochrome c release in a cell.
  • a neurological disorder e.g. Alzheimer's disease, or ischemia
  • a control level of cytochrome c release may be a predetermined value, which can take a variety of forms. It can be a single value, such as a median or mean. It can be established based upon comparative groups, such as in disease-free groups that have normal levels of cytochrome c release. Other comparative groups may be groups of subjects with specific neurological disorders, e.g.
  • disease-free cells and/or tissues maybe used as comparative groups for cells or tissues that have a cytochrome c release-associated disorder.
  • a compound that inhibits and thereby reduces the level of cytochrome c released is an agent that reduces a cell death-associated disease or disorder.
  • the level of cytochrome c release may be one that is below the level seen in subjects with a neurological disorder, e.g. may be a level that is clinically asymptomatic.
  • the invention relates in part to the administration of a cytochrome c release-inhibiting compound of the invention in an amount effective to treat or prevent cytochrome c release in cells, tissues, and/or subjects with a cytochrome c release-associated disease or disorder.
  • the cytochrome c release-inhibiting compounds include functional analogs, derivatives, and/or variants of the cytochrome c release-inhibiting compounds of the invention.
  • the term-cytochrome c release-inhibiting compounds may include functional analogs, derivatives, and/or variants of the calcimycin, dipyrone, parthenolide, hydroxyprogesterone caproate, mephenytoin, N-acetyl-DL-tryptophan, fendiline hydrochloride, ritanserin, azathioprine, bepridil hydrochloride, probucol, methoazolamide, doxycycline hydrochloride, melatonin, and/or minocycline hydrochloride compounds set forth herein.
  • cytochrome c release-inhibiting compounds of Table 1 may be made to enhance a property of a compound, such as stability.
  • Functional analogs, derivatives, and variants of the compounds of Table 1 may also be made to provide a novel activity or property to a compound of Table 1, for example, to enhance detection, to enhance potency, to reduce side effects, etc.
  • modifications to a cytochrome c release-inhibiting compound of the invention can be made to the structure or side groups of the compound and can include deletions, substitutions, and additions of atoms, or side groups.
  • modifications can be made by addition of a linker molecule, addition of a detectable moiety, such as biotin or a fluorophore, chromophore, enzymatic, and/or radioactive label, and the like.
  • an analog of a molecule may have a higher level of cytochrome c release-inhibition activity than the original compound.
  • Chemical groups that can be added to or substituted in the molecules include: hydrido, alkyl, alkenyl, alkynyl, cycloalkyl, heterocycyl, aryl, heteroaryl, alkoxy, aryloxy, sulfoxy, acyl, amino, acyloxy, acylamino, carboalkoxy, carboxyamido, carboxyamido, halo and thio groups. Substitutions can replace one or more chemical groups or atoms on the molecules.
  • acyl is defined as a carbonyl radical attached to an alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl or heteroaryl group, examples of such radicals being acetyl and benzoyl.
  • amino denotes a nitrogen radical containing two substituents independently selected from the group consisting of hydrido, alkyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl.
  • acyloxy denotes an oxygen radical adjacent to an acyl group.
  • acylamino denotes a nitrogen radical adjacent to an acyl group.
  • carboalkoxy is defined as a carbonyl radical adjacent to an alkoxy or aryloxy group.
  • carboxyamido denotes a carbonyl radical adjacent to an amino group.
  • carbboxy embraces a carbonyl radical adjacent to an alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl or heteroaryl group.
  • halo is defined as a bromo, chloro, fluoro or iodo radical.
  • thio denotes a radical containing a substituent group independently selected from hydrido, alkyl, cycloalkyl, heterocyclyl, aryl and heteroaryl, attached to a divalent sulfur atom, such as, methylthio and phenylthio.
  • alkyl is defined as a linear or branched, saturated radical having one to about ten carbon atoms unless otherwise specified. Preferred alkyl radicals are "lower alkyl” radicals having one to about five carbon atoms.
  • One or more hydrogen atoms can also be replaced by a substituent group selected from acyl, amino, acylamino, acyloxy, carboalkoxy, carboxy, carboxyamido, cyano, halo, hydroxy, nitro, thio, alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, alkoxy, aryloxy, sulfoxy, and formyl.
  • alkyl groups include methyl, tert-butyl, isopropyl, and methoxymethyl.
  • alkenyl embraces linear or branched radicals having two to about twenty carbon atoms, preferably three to about ten carbon atoms, and containing at least one carbon-carbon double bond.
  • One or more hydrogen atoms can also be replaced by a substituent group selected from acyl, amino, acylamino, acyloxy, carboalkoxy, carboxy, carboxyamido, cyano, halo, hydroxy, nitro, thio, alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, alkoxy, aryloxy, sulfoxy, and formyl.
  • alkenyl groups include ethylenyl or phenyl ethylenyl.
  • alkynyl denotes linear or branched radicals having from two to about ten carbon atoms, and containing at least one carbon-carbon triple bond.
  • One or more hydrogen atoms can also be replaced by a substituent group selected from acyl, amino, acylamino, acyloxy, carboalkoxy, carboxy, carboxyamido, cyano, halo, hydroxy, nitro, thio, alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, alkoxy, aryloxy, sulfoxy, and formyl.
  • alkynyl groups include propynyl.
  • aryl denotes aromatic radicals in a single or fused carbocyclic ring system, having from five to twelve ring members.
  • One or more hydrogen atoms may also be replaced by a substituent group selected from acyl, amino, acylamino, acyloxy, carboalkoxy, carboxy, carboxyamido, cyano, halo, hydroxy, nitro, thio, alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, alkoxy, aryloxy, sulfoxy, and formyl.
  • substituent group selected from acyl, amino, acylamino, acyloxy, carboalkoxy, carboxy, carboxyamido, cyano, halo, hydroxy, nitro, thio, alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, alkoxy
  • Heteroaryl embraces aromatic radicals which contain one to four hetero atoms selected from oxygen, nitrogen and sulfur in a single or fused heterocyclic ring system, having from five to fifteen ring members.
  • One or more hydrogen atoms may also be replaced by a substituent group selected from acyl, amino, acylamino, acyloxy, carboalkoxy, carboxy, carboxyamido, cyano, halo, hydroxy, nitro, thio, alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, alkoxy, aryloxy, sulfoxy, and formyl.
  • heteroaryl groups include, pyridinyl, thiazolyl, thiadiazoyl, isoquinolinyl, pyrazolyl, oxazolyl, oxadiazoyl, triazolyl, and pyrrolyl groups.
  • cycloalkyl is defined as a saturated or partially unsaturated carbocyclic ring in a single or fused carbocyclic ring system having from three to twelve ring members.
  • One or more hydrogen atoms may also be replaced by a substituent group selected from acyl, amino, acylamino, acyloxy, carboalkoxy, carboxy, carboxyamido, cyano, halo, hydroxy, nitro, thio, alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, alkoxy, aryloxy, sulfoxy, and formyl.
  • Examples of a cycloalkyl group include cyclopropyl, cyclobutyl, cyclohexyl, and cycloheptyl.
  • heterocyclyl embraces a saturated or partially unsaturated ring containing zero to four hetero atoms selected from oxygen, nitrogen and sulfur in a single or fused heterocyclic ring system having from three to twelve ring members.
  • One or more hydrogen atoms may also be replaced by a substituent group selected from acyl, amino, acylamino, acyloxy, carboalkoxy, carboxy, carboxyamido, cyano, halo, hydroxy, nitro, thio, alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, alkoxy, aryloxy, sulfoxy, and formyl.
  • heterocyclyl group examples include morpholinyl, piperidinyl, and pyrrolidinyl.
  • alkoxy denotes oxy-contaming radicals substituted with an alkyl, cycloalkyl or heterocyclyl group. Examples include methoxy, i"er/-butoxy, benzyloxy and cyclohexyloxy.
  • aryloxy denotes oxy-containing radicals substituted with an aryl or heteroaryl group. Examples include phenoxy.
  • sulfoxy is defined as a hexavalent sulfur radical bound to two or three substituents selected from the group consisting of oxo, alkyl, cycloalkyl, heterocyclyl, aryl and heteroaryl, wherein at least one of said substituents is oxo.
  • the cytochrome c release-inhibiting compounds of the invention also include, but are not limited to any pharmaceutically acceptable salts, esters, or salts of an ester of each compound.
  • salts that may be used, which is not intended to be limiting include: chloride, acetate, hydrochloride, methansulfonate or other salt of a compound of Table 1 or a functional analog, derivative, variant, or fragment of the compound.
  • Derivatives of the compounds of Table 1 include compounds which, upon administration to a subject in need of such administration, deliver (directly or indirectly) a pharmaceutically active cytochrome c release-inhibiting compound as described herein.
  • An example of pharmaceutically active derivatives of the invention includes, but is not limited to, pro-drugs.
  • a pro-drug is a derivative of a compound that contains an additional moiety that is susceptible to removal in vivo yielding the parent molecule as a pharmacologically active agent.
  • An example of a pro-drug is an ester that is cleaved in vivo to yield a compound of interest.
  • Pro-drugs of a variety of compounds, and materials and methods for derivatizing the parent compounds to create the pro-drugs are known to those of ordinary skill in the art and may be adapted to the present invention.
  • Analogs, variants, and derivatives of the compounds of the invention set forth in Table 1 may be identified using standard methods known to those of ordinary skill in the art. Useful methods involve identification of compounds having similar chemical structure, similar active groups, chemical family relatedness, and other standard characteristics.
  • the chemical elements are identified in accordance with the Periodic Table of the Elements, CAS version, Handbook of Chemistry and Physics 75 th Ed., inside cover, and specific functional groups are defined as described therein. Additionally, general principles of organic chemistry, as well as specific functional moieties and reactivity, are described in "Organic Chemistry", Thomas Sorrell, University Science Books, Sausalito. 1999, the contents of which are incorporated herein by reference in their entirety.
  • the invention also involves methods for determining the functional activity of cytochrome c release-inhibiting compounds described herein.
  • the function or status of a compound as a cytochrome c release-inhibiting compound can be determined according to assays known as described herein. For example, cells can be contacted with a candidate cytochrome c release-inhibiting compound under conditions that produce cytochrome release, and standard procedures can be used to determine whether cytochrome c release activity is modulated (inhibited or enhanced) by the compound and/or whether cell death levels are modulated by the compound. Such methods may also be utilized to determine the status of analogs, variants, and derivatives as inhibitors of cytochrome c release.
  • cytochrome c release-inhibiting compound examples of methods with which the ability of a cytochrome c release-inhibiting compound to inhibit cytochrome c release and/or to inhibit cell death can be tested, are in vitro and in vivo assay systems provided herein in the Examples section.
  • the level of cytochrome c release can be measured in the system both before and after contacting the system with a candidate cytochrome c release-inhibitor compound as an indication of the effect of the compound on the level of cytochrome c release.
  • Secondary screens may further be used to verify the compounds identified as inhibitors of cytochrome c release and/or cell death.
  • derivatives, analogs, and variants of cytochrome c release-inhibiting compounds can be tested for their cytochrome c release-inhibition activity and/or cell death inhibiting activity by using an activity assay (see examples).
  • An example of an assay method is contacting a tissue or cell sample with a cytochrome c release-inhibitor compound and determining the compound's inhibitory activity as described herein. Contacting a similar cell or tissue sample with an analog of the cytochrome c release-inhibiting compound, determining its activity, and then comparing the two activity results as a measure of the efficacy of the analog's cytochrome c release-inhibiting activity.
  • an in vivo assay may be used to. determine the functional activity of cytochrome c release-inhibiting compounds described herein.
  • animal models of cytochrome c release-associated disease or disorder can be treated with a cytochrome c release-inhibiting compound of the invention.
  • Cytochrome c release-inhibiting activity and/or cell death may be assayed using methods described herein, which may include labeling or imaging methods.
  • animals with and without cytochrome c release-inhibiting compound treatment can be examined for behavior and/or survival as an indication of the effectiveness and/or efficacy of the compounds.
  • Behavior may be assessed by examination of symptoms of aberrant cytochrome c release as described herein.
  • aberrant means abnormal.
  • test and control animals may be examined following administration of a cytochrome c release-inhibiting compound of the invention.
  • test animals are administered a cytochrome c release-inhibiting compound of the invention and control animals are not. Any resulting change in cytochrome c release and/or cell death can then be determined for each type of animal using known methods in the art as described herein.
  • Such assays may be used to compare levels of cytochrome c release and/or cell death in animals administered the candidate cytochrome c release-inhibiting compound to control levels of cytochrome c release and/or cell death in animals not administered the cytochrome c release-inhibiting compound as an indication that the putative cytochrome c release-inhibiting compound is effective to reduce cytochrome c release and/or cell death.
  • a candidate cytochrome c release-inhibiting compound may be administered to both a test and control animal and the results on cytochrome c release and/or cell death may be compared as a measure of the efficacy of the compound.
  • cytochrome c release-inhibiting compounds are verified as inhibiting cytochrome c release using art-known assays or assays as described herein (e.g., in Examples), further biochemical and molecular techniques may be used to identify the targets of these compounds and to elucidate the specific roles that these target molecules play in the process of cytochrome c release and/or cell death in associated diseases and/or disorders.
  • a cytochrome c release-inhibiting compound of the invention maybe delivered to the cell using standard methods known to those of ordinary skill in the art. Various techniques may be employed for introducing cytochrome c release-inhibiting compounds of the invention to cells, depending on whether the compounds are introduced in vitro or in vivo in a host.
  • the cytochrome c release-inhibiting compounds (also referred to herein as therapeutic compounds and/or pharmaceutical compounds) of the present invention are administered in pharmaceutically acceptable preparations.
  • Such preparations may routinely contain pharmaceutically acceptable concentrations of salt, buffering agents, preservatives, compatible carriers, and optionally other therapeutic agents.
  • pharmaceutically acceptable means a non-toxic material that does not interfere with the effectiveness of the biological activity of the active ingredients.
  • the characteristics of the carrier will depend on the route of administration.
  • the therapeutics of the invention can be administered by any conventional route, including injection or by gradual infusion over time.
  • the administration may, for example, be oral, intravenous, intraperitoneal, intrathecal, intramuscular, intranasal, intracavity, subcutaneous, intradermal, mucosal, transdermal, or transdermal.
  • compositions suitable for parenteral administration conveniently comprise a sterile aqueous preparation of the therapeutic agent, which is preferably isotonic with the blood of the recipient. This aqueous preparation may be formulated according to known methods using those suitable dispersing or wetting agents and suspending agents.
  • the sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally-acceptable diluent or solvent, for example as a solution in 1, 3-butane diol.
  • a non-toxic parenterally-acceptable diluent or solvent for example as a solution in 1, 3-butane diol.
  • acceptable vehicles and solvents that may be employed are water, Ringer's solution, and isotonic sodium chloride solution.
  • sterile, fixed oils are conventionally employed as a solvent or suspending medium.
  • any bland fixed oil may be employed including synthetic mono or di-glycerides.
  • fatty acids such as oleic acid find use in the preparation of injectables.
  • Carrier formulations suitable for oral, subcutaneous, intravenous, intramuscular, etc. can be found in Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, PA.
  • compositions suitable for oral administration may be presented as discrete units such as capsules, cachets, tablets, or lozenges, each containing a predetermined amount of the therapeutic agent.
  • Other compositions include suspensions in aqueous liquors or non-aqueous liquids such as a syrup, an elixir, or an emulsion.
  • a cytochrome c release-inhibiting compound of the invention may be delivered in the form of a delivery complex.
  • the delivery complex may deliver the cytochrome c release-inhibiting compound into any cell type, or may be associated with a molecule for targeting a specific cell type.
  • Examples of delivery complexes include a cytochrome c release-inhibiting compound of the invention associated with: a sterol (e.g., cholesterol), a lipid (e.g., a cationic lipid, virosome or liposome), or a target cell specific binding agent (e.g., an antibody, including but not limited to monoclonal antibodies, or a ligand recognized by target cell specific receptor):
  • a sterol e.g., cholesterol
  • a lipid e.g., a cationic lipid, virosome or liposome
  • a target cell specific binding agent e.g., an antibody, including but not limited to monoclonal antibodies, or a ligand recognized by target cell specific receptor
  • liposomes to deliver a cytochrome c release-inhibiting compound of the invention into a cell.
  • Liposomes may be targeted to a particular tissue, such neuronal cells, (e.g. hippocampal cells, etc), or other cell type, by coupling the liposome to a specific ligand such as a monoclonal antibody, sugar, glycolipid, or protein.
  • a specific ligand such as a monoclonal antibody, sugar, glycolipid, or protein.
  • proteins include proteins or fragments thereof specific for a particular cell type, antibodies for proteins that undergo internalization in cycling, proteins that target intracellular localization and enhance intracellular half life, and the like.
  • a vehicle e.g. a liposome
  • a cytochrome c release-inhibiting compound of the invention may be desirable to target the compound to particular cells, for example specific neuronal cells, including specific tissue cell types, e.g. hippocampal cells or other tissue-specific nervous system cells.
  • a cytochrome c release-inhibiting compound may be desirable to target a cytochrome c release-inhibiting compound to another cell type, including, but not limited to, liver cells, cardiac cells, vascular cells, etc.
  • a vehicle e.g. a liposome
  • a neuronal cell, liver cell, etc. may have a targeting molecule attached thereto that is an antibody specific for a surface membrane polypeptide of the cell type or may have attached thereto a ligand for a receptor on the cell type.
  • a targeting molecule can be bound to or incorporated within the cytochrome c release-inhibiting compound delivery vehicle.
  • proteins that bind to a surface membrane protein associated with endocytosis may be incorporated into the liposome formulation for targeting and/or to facilitate uptake.
  • Liposomes are commercially available from Invitrogen, for example, as LIPOFECTINTM and LIPOFECTACETM, which are formed of cationic lipids such as N-[l-(2,3 dioleyloxy)-propyl]-N, N, N-trimethylammonium chloride (DOTMA) and dimethyl dioctadecylammonium bromide (DDAB).
  • LIPOFECTINTM LIPOFECTINTM
  • LIPOFECTACETM LIPOFECTINTM and LIPOFECTACETM, which are formed of cationic lipids such as N-[l-(2,3 dioleyloxy)-propyl]-N, N, N-trimethylammonium chloride (DOTMA) and dimethyl dioctadecylammonium bromide (DDAB).
  • DOTMA N-[l-(2,3 dioleyloxy)-propyl]-N, N, N-trimethylammonium chloride
  • DDAB di
  • the invention provides a composition of the above-described agents for use as a medicament, methods for preparing the medicament and methods for the sustained release of the medicament in vivo.
  • Delivery systems can include time-release, delayed release or sustained release delivery systems. Such systems can avoid repeated administrations of the therapeutic agent of the invention, increasing convenience to the subject and the physician.
  • Many types of release delivery systems are available and known to those of ordinary skill in the art. They include, but are not limited to, polymer-based systems such as polylactic and polyglycolic acid, poly(lactide-glycolide), copolyoxalates, polyanhydrides, polyesteramides, polyorthoesters, polyhydroxybutyric acid, and polycaprolactone.
  • Microcapsules of the foregoing polymers containing drugs are described in, for example, U.S. Pat. No. 5,075,109.
  • Nonpolymer systems that are lipids including sterols such as cholesterol, cholesterol esters and fatty acids or neutral fats such as mono-, di- and tri-glycerides; phospholipids; hydrogel release systems; silastic systems; peptide based systems; wax coatings, compressed tablets using conventional binders and excipients, partially fused implants and the like.
  • Specific examples include, but are not limited to: (a) erosional systems in which the polysaccharide is contained in a form within a matrix, found in U.S. Patent Nos.
  • the preferred vehicle is a biocompatible microparticle or implant that is suitable for implantation into the mammalian recipient. Exemplary bioerodible implants that are useful in accordance with this method are described in PCT International application no.
  • WO 95/24929 entitled “Polymeric Gene Delivery System” describes a biocompatible, preferably biodegradable polymeric matrix for containing an exogenous gene under the control of an appropriate promoter.
  • the polymeric matrix is used to achieve sustained release of the exogenous gene in the patient.
  • the compound(s) of the invention is encapsulated or dispersed within the biocompatible, preferably biodegradable polymeric matrix disclosed in WO 95/24929.
  • the polymeric matrix preferably is in the form of a microparticle such as a microsphere (wherein the compound is dispersed throughout a solid polymeric matrix) or a microcapsule (wherein the compound is stored in the core of a polymeric shell).
  • polymeric matrix for containing the compounds of the invention include films, coatings, gels, implants, and stents.
  • the size and composition of the polymeric matrix device is selected to result in favorable release kinetics in the tissue into which the matrix device is implanted.
  • the size of the polymeric matrix device further is selected according to the method of delivery which is to be used.
  • the polymeric matrix composition can be selected to have both favorable degradation rates and also to be formed of a material which is bioadhesive, to further increase the effectiveness of transfer when the device is administered to a vascular surface.
  • the matrix composition also can be selected not to degrade, but rather, to release by diffusion over an extended period of time.
  • Both non-biodegradable and biodegradable polymeric matrices can be used to deliver agents and compounds of the invention of the invention to the subject.
  • Biodegradable matrices are preferred.
  • Such polymers may be natural or synthetic polymers. Synthetic polymers are preferred.
  • the polymer is selected based on the period of time over which release is desired, generally in the order of a few hours to a year or longer. Typically, release over a period ranging from between a few hours and three to twelve months is most desirable.
  • the polymer optionally is in the form of a hydrogel that can absorb up to about 90% of its weight in water and further, optionally is cross-linked with multi-valent ions or other polymers.
  • the agents and/or compounds of the invention are delivered using the bioerodible implant by way of diffusion, or more preferably, by degradation of the polymeric matrix.
  • exemplary synthetic polymers which can be used to form the biodegradable delivery system include: polyamides, polycarbonates, polyalkylenes, polyalkylene glycols, polyalkylene oxides, polyalkylene terepthalates, polyvinyl alcohols, polyvinyl ethers, polyvinyl esters, polyvinyl halides, polyvinylpyrrolidone, polyglycolides, polysiloxanes, polyurethanes and co-polymers thereof, alkyl cellulose, hydroxyalkyl celluloses, cellulose ethers, cellulose esters, nitro celluloses, polymers of acrylic and methacrylic esters, methyl cellulose, ethyl cellulose, hydroxypropyl cellulose, hydroxy-propyl methyl cellulose, hydroxybutyl methyl cellulose, cellulose acetate
  • non-biodegradable polymers examples include ethylene vinyl acetate, poly(meth)acrylic acid, polyamides, copolymers and mixtures thereof.
  • biodegradable polymers include synthetic polymers such as polymers of lactic acid and glycolic acid, polyanhydrides, poly(ortho)esters, polyurethanes, poly(butic acid), poly(valeric acid), and poly(lactide-cocaprolactone), and natural polymers such as alginate and other polysaccharides including dextran and cellulose, collagen, chemical derivatives thereof (substitutions, additions of chemical groups, for example, alkyl, alkylene, hydroxylations, oxidations, and other modifications routinely made by those skilled in the art), albumin and other hydrophilic proteins, zein and other prolamines and hydrophobic proteins, copolymers and mixtures thereof, m general, these materials degrade either by enzymatic hydrolysis or exposure to water in vivo, by surface or bulk erosion.
  • synthetic polymers such as polymers of lactic acid and glycolic acid, polyanhydrides, poly(ortho)esters, polyurethanes, poly(butic acid), poly(val
  • Bioadhesive polymers of particular interest include bioerodible hydrogels described by H. S. Sawhney, C. P. Pathak and J. A. Hubell in Macromolecules, 1993, 26, 581-587, the teachings of which are incorporated herein by reference, polyhyaluronic acids, casein, gelatin, glutin, polyanhydrides, polyacrylic acid, alginate, chitosan, poly(methyl methacrylates), poly(ethyl methacrylates), poly(butylmethacrylate), poly(isobutyl methacrylate), poly(hexylmethacrylate), poly(isodecyl methacrylate), poly(lauryl methacrylate), poly(phenyl methacrylate), poly(methyl acrylate), poly(isopropyl acrylate), poly(isobutyl acrylate), and poly(octadecyl acrylate).
  • long-term sustained release implant may be particularly suitable for treatment of subjects with an established neurological disorder or other cell death-associated condition as well as subjects at risk of developing a neurological disorder, or other cell death-associated condition.
  • Long-term release means that the implant is constructed and arranged to deliver therapeutic levels of the active ingredient for at least 7 days, and preferably 30-60 days, and most preferably months or years.
  • the implant may be positioned at or near the site of the neurological damage or the area of the brain or nervous system affected by or involved in the neurological disorder.
  • Long-term release implants may also be used in non-neuronal tissues and organs to allow regional administration of a cytochrome a release- inhibiting compound of the invention. Long-term sustained release implants are well known to those of ordinary skill in the art and include some of the release systems described above.
  • Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions.
  • non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate.
  • Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media.
  • Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's or fixed oils.
  • Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose), and the like. Preservatives and other additives may also be present such as, for example, antimicrobials, anti-oxidants, chelating agents, and inert gases and the like.
  • the preparations of the invention are administered in effective amounts.
  • An effective amount is that amount of a pharmaceutical preparation that alone, or together with further doses, stimulates the desired response.
  • the desired response is reducing the onset, stage or progression of the aberrant cytochrome release and/or cytochrome c release-associated cell death. This may involve only slowing the progression of the damage temporarily, although more preferably, it involves halting the progression of the damage permanently.
  • An effective amount for preventing and/or treating aberrant cytochrome c release is that amount that reduces the amount or level of cytochrome c release, when the cell or subject is a cell or subject with a cytochrome c release-associated disease or disorder, with respect to that amount that would occur in the absence of the active compound.
  • the invention involves, in part, the administration of an effective amount of a cytochrome c release-inhibiting compound of the invention.
  • the cytochrome c release-inhibiting compounds of the invention are administered in effective amounts.
  • effective amounts of a cytochrome c release-inhibiting compound will be determined in clinical trials, establishing an effective dose for a test population versus a control population in a blind study.
  • an effective amount will be that amount that diminishes or eliminates a cytochrome c release-associated disease or disorder and/or a cell death-associated disease or disorder in a cell, tissue, and/or subject.
  • an effective amount may be the amount that when administered reduces the amount of cell death from the amount that would occur in the subject or tissue without the administration of the cytochrome c release-inhibiting compound of the invention.
  • the pharmaceutical compound dosage may be adjusted by the individual physician or veterinarian, particularly in the event of any complication.
  • a therapeutically effective amount typically varies from 0.01 mg/kg to about 1000 mg/kg, preferably from about 0.1 mg/kg to about 200 mg/kg, and most preferably from about 0.2 mg/kg to about 20 mg/kg, in one or more dose administrations daily, for one or more days. It will be recognized by those of skill in the art that some of the cytochrome c release-inhibiting compounds may have detrimental effects at high amounts. Thus, an effective amount for use in the methods of the invention may be optimized such that the amount administered results in minimal negative side effects and maximum cytochrome c inhibition.
  • the absolute amount will depend upon a variety of factors, including the material selected for administration, whether the administration is in single or multiple doses, and individual subject parameters including age, physical condition, size, weight, and the stage of the disease or disorder. These factors are well known to those of ordinary skill in the art and can be addressed with no more than routine experimentation.
  • the pharmaceutical compounds of the invention maybe administered alone, in combination with each other, and/or in combination with other drug therapies that are administered to subjects with cell death-associated diseases and/or disorders.
  • Additional drug therapies (for treatment and/or prophylaxis) that may be administered with pharmaceutical compounds of the invention include, art-known methods of treating cell death-associated diseases and/or disorders and/or cytochrome c release-associated diseases and/or disorders.
  • Alternative drug therapies are known to those of ordinary skill in the art and are administered by modes known to those of skill in the art.
  • the drug therapies are administered in amounts that are effective to achieve the physiological goals (to reduce symptoms and damage from cytochrome c release-associated disease or disorder in a subject, e.g. cell death), in combination with the pharmaceutical compounds of the invention.
  • the alternative drug therapies may be administered in amounts which are not capable of preventing or reducing the physiological consequences of the cytochrome c release-associated disease and/or disorder when the drug therapies are administered alone, but which are capable of preventing or reducing the physiological consequences of a cytochrome c release-associated disease and/or disorder when administered in combination with one or more cytochrome c release-inhibiting compounds of the invention.
  • Diagnostic tests known to those of ordinary skill in the art may be used to assess the level of cytochrome c release and/or the level of cell death in a subject and to evaluate a therapeutically effective amount of a pharmaceutical compound administered. Examples of diagnostic tests are set forth below.
  • a first determination of cytochrome c release in a cell and/or tissue may be obtained using one of the methods described herein (or other methods known in the art), and a second, subsequent determination of the level of cytochrome c release may be done.
  • a comparison of the cytochrome c release activity and/or the level of cell death at the different time points maybe used to assess the effectiveness of administration of a pharmaceutical compound of the invention as a prophylactic or a treatment of the cytochrome c release-associated disease or disorder and/or a cell death-associated disease or disorder.
  • Family history or prior occurrence of a cytochrome c release-associated disease or disorder even if the cytochrome c release-associated disease or disorder is absent in a subject at present, may be an indication for prophylactic intervention by administering a pharmaceutical compound described herein to reduce or prevent aberrant cytochrome c release.
  • An example of a method of diagnosis of aberrant cytochrome c release that can be performed using standard methods such as, but not limited to: imaging methods, electrophysiological methods, and histological methods. Additional methods of diagnosis and assessment of cytochrome c release-associated disease or disorders and the resulting cell death or damage are known to those of skill in the art.
  • cytochrome c release-associated diseases and/or disorders can be monitored for assessment of cytochrome c release following onset of a cytochrome c release-associated disease or disorder.
  • These features include, but are not limited to: assessment of the presence of cell death, neuronal cell lesions, spinal cord lesions, brain lesions, organ lesions, and behavioral abnormalities.
  • Such assessment can be done with methods known to one of ordinary skill in the art, such as behavioral testing and imaging studies, such as radiologic studies, CT scans, PET scans, etc.
  • the invention also provides a pharmaceutical kit comprising one or more containers comprising one or more of the cytochrome c release-inhibiting compounds of the invention and/or formulations of the invention.
  • the kit may also include instructions for the use of the one or more cytochrome c release-inhibiting compounds or formulations of the invention for the treatment of a cytochrome c release-associated disease or disorder.
  • the kits of the invention may also comprise additional drugs for preventing and/or treating a cytochrome c release-associated disease or disorder.
  • the invention further provides efficient methods of identifying pharmacological agents or lead compounds for agents and compounds that inhibit (or enhance) cytochrome c release.
  • the screening methods involve assaying for compounds which modulate (enhance or inhibit) the level of cytochrome c release.
  • the screening methods may measure the level of cytochrome c release directly, e.g., screening methods described herein.
  • screening methods may be utilized that measure a secondary effect of cytochrome c release, or example, the level of cell death in a cell or tissue sample or by measuring behavioral characteristics of the cytochrome c release-associated disease.
  • assays for pharmacological agents can be used in accordance with this aspect of the invention, including, cytochrome c release assays, cell viability assays, cell-free assays, cell-based assays, etc.
  • the term "pharmacological agent” means cytochrome c release-inhibiting compounds.
  • An example of such an assay that is useful to test candidate cytochrome c release-inhibiting compounds is provided in the Examples section.
  • the assay mixture comprises a candidate pharmacological agent.
  • a plurality of assay mixtures is run in parallel with different agent concentrations to obtain a different response to the various concentrations.
  • one of these concentrations serves as a negative control, i.e., at zero concentration of agent or at a concentration of agent below the limits of assay detection.
  • Candidate compounds useful in accordance with the invention encompass numerous chemical classes, although typically they are organic compounds.
  • the candidate pharmacological agents are small organic compounds, i.e., those having a molecular weight of more than 50 yet less than about 2500, preferably less than about 1000 and, more preferably, less than about 500.
  • Candidate compounds comprise functional chemical groups necessary for structural interactions with proteins and/or nucleic acid molecules.
  • the candidate compounds can comprise cyclic carbon or heterocyclic structure and/or aromatic or polyaromatic structures substituted with one or more of the above-identified functional groups.
  • Candidate compounds also can be biomolecules such as peptides, saccharides, fatty acids, sterols, isoprenoids, purines, pyrimidines, derivatives or structural analogs of the above, or combinations thereof and the like.
  • the agent typically is a DNA or RNA molecule, although modified nucleic acid molecules are also contemplated.
  • cell-based assays as described herein can be performed using cell samples and/or cultured cells.
  • Biopsy cells and tissues as well as cell lines grown in culture are useful in the methods of the invention.
  • Candidate compounds are obtained from a wide variety of sources including libraries of synthetic or natural compounds. For example, numerous means are available for random and directed synthesis of a wide variety of organic compounds and biomolecules, including expression of randomized oligonucleotides, synthetic organic combinatorial libraries, phage display libraries of random peptides, and the like.
  • libraries of natural compounds in the form of bacterial, fungal, plant and animal extracts are available or readily produced.
  • natural and synthetically produced libraries and compounds can be readily be modified through conventional chemical, physical, and biochemical means.
  • pharmacological compounds may be subjected to directed or random chemical modifications such as acylation, alkylation, esterification, amidification, etc. to produce structural analogs of the compounds.
  • Candidate compounds also include analogs, derivatives, and/or variants of the cytochrome c release-inhibiting compounds described herein.
  • reagents such as salts, buffers, neutral proteins (e.g., albumin), detergents, etc. which may be used to facilitate optimal binding, or to reduce non-specific or background interactions of the reaction components.
  • reagents such as salts, buffers, neutral proteins (e.g., albumin), detergents, etc. which may be used to facilitate optimal binding, or to reduce non-specific or background interactions of the reaction components.
  • Other reagents that improve the efficiency of the assay such as protease inhibitors, nuclease inhibitors, antimicrobial agents, and the like may also be used.
  • cytochrome c release-inhibitor assay is described herein, which may be used to identify candidate compounds that inhibit cytochrome c release and/or cell death.
  • the mixture of the foregoing assay materials is incubated under conditions whereby, but for the presence of the candidate pharmacological compound, there is an above-normal level of cytochrome c release and/or cell death.
  • the order of addition of components, incubation temperature, time of incubation, and other parameters of the assay may be readily determined. Such experimentation merely involves optimization of the assay parameters, not the fundamental composition of the assay. Incubation temperatures typically are between 4 0 C and 4O 0 C. Incubation times preferably are minimized to facilitate rapid, high throughput screening, and typically are between 0.1 and 10 hours.
  • the level of cytochrome c release may be detected by any convenient method available to the user.
  • an ELISA assay may be used for detection.
  • Detection may be effected in any convenient way for cell-based assays.
  • one of the components usually comprises, or is coupled to, a detectable label.
  • labels can be used, such as those that provide direct detection (e.g., radioactivity, luminescence, optical or electron density, etc.) or indirect detection (e.g., epitope tag such as the FLAG epitope, enzyme tag such as horse-radish peroxidase, etc.).
  • Labels may be directly detected through optical or electron density, radioactive emissions, nonradiative energy transfers, etc. or indirectly detected with antibody conjugates, strepavidin-biotin conjugates, etc. Methods for detecting the labels are well known in the art.
  • cytochrome c release from the mitochondria may support a novel therapeutic approach to neurodegeneration
  • Inhibitors of cytochrome c release were identified from a library of 1,040 drugs approved by the Food and Drug Administration. Effective drugs at the level of purified mitochondria were evaluated in several paradigms of neuronal cell death. Two compounds, methazolamide and melatonin, were evaluated in further depth in cellular and in vivo models of neuronal injury. We demonstrated that these compounds prevented neuronal cell death by blocking the dissipation of mitochondrial membrane potential ( ⁇ m).
  • methazolamide and melatonin reduced infarct size and decreased neurologic deficit in a mouse model of cerebral ischemia.
  • Our findings demonstrate the utility of a mitochondrial screen for the identification of inhibitors of cell death. Given that these are FDA approved drugs, and many of them effectively cross the blood brain, these drugs may expeditiously progress into human trials of neurologic diseases featuring caspase-mediated cell death.
  • Minocycline a broadly neuroprotective drug, acts at least in part by directly inhibiting the release of cytochrome c from mitochondria (Shan, 2002).
  • Minocycline is protective in experimental models of stroke, traumatic brain and spinal cord injury, HD, ALS, Parkinson disease, and multiple sclerosis (Brundula et al., 2002; Chen et al., 2000; Friedlander, 2003; Wang et al., 2003; Wu et al., 2002; Yrjanheikki et al., 1998; Zhu et al., 2002).
  • the drugs were obtained from the 1,040 compounds National Institute of Neurological
  • the cell line ST14A are striatal neurons conditionally immortalized by transfection with a temperature-sensitive form of the SV40 large T-antigen.
  • N548mu [ntl955-128] stable huntingtin ST14A (mutant-htt ST14A) cells were cultured as described (Rigamonti et al., 2000). Mutant-htt ST14A cells were kindly provided by Dr. Maria Cattaneo (University of Milan, Italy). Cells were shifted to the non-permissive temperature of 37°C in serum-deprived medium (SDM) for the indicated time with or without different concentrations of methazolamide, melatonin, minocycline, or gossypol-acetic acid.
  • SDM serum-deprived medium
  • PCN primary cortical neurons
  • E 14-E 16 Culture of primary cortical neurons (PCN) (E 14-E 16) was performed as previously described (Zhang et al . , 2003b).
  • PCN were stimulated with 1 mM H2O2 or 500 ⁇ M NMDA for 18-20 h, or OGD as described previously (Zhang et al., 2003b).
  • the culture medium was replaced with glucose-free Earle's balanced salt solution with or without different concentrations of test drugs, and the cells were placed in an anaerobic chamber with BBL GasPak Plus (Becton Dickinson), which reduces the oxygen concentration to ⁇ 100 ppm within 90 min.
  • Control cells were incubated in Earle's balanced salt solution with glucose in a normoxic incubator for the same period. After 3 h, OGD was terminated by a return to normal culture conditions. All experiments used 2 h of pre-incubation with test drugs. Cell death was determined by MTS or LDH assay.
  • LDH assay Primary cortical neuron cell death was determined by the LDH assay according to the manufacturer's instructions (Hoffinann-La Roche Inc. (Roche), Nutley, N.J.,) (Zhang et al., 2003b).
  • Enzyme-linked immunosorbent assay The NINDS library was obtained as 13 kits with 80 compounds in each. A single 96- well ELISA plate was dedicated to each kit of 80 compounds. Drugs were assayed with a separate mitochondrial preparation for each kit. For each sample within a given 96-well plate, the absorbance at 450 nm (a measure of cytochrome c concentration) was corrected for imperfections in the plastic by subtracting the reading at 540 nm. Cytochrome c concentration was calculated by interpolating these values on a standard curve constructed for each plate.
  • mPT mitochondrial permeability transition
  • Mitochondria swelling was induced by Ca 2+ , tert-butylhydroperoxide (tBH), PhAsO, or diamide and monitored in standard spectroscopic assay at 540 ran. Methazolamide and melatonin were respectively applied to mitochondria.
  • MCAO Permanent middle cerebral artery occlusion
  • Focal cerebral ischemia was produced as described previously (Zhang et al., 2003b). Briefly, anesthesia was induced in 6- to 8-week-old C57BL/6J mice (Jackson Laboratory, Bar Harbor, ME) with 2% (vol/vol) isoflurane (70% N 2 O/30% O 2 ) and was maintained with a 1-0.5% concentration. Rectal temperature was maintained between 37.0 and 37.5°C with a heating pad (Harvard Apparatus, Hamden, CT).
  • Focal cerebral ischemia was induced by an intraluminal 7-0 nylon thread with a silicone tip (180 ⁇ m diameter) introduced into the right cervical internal carotid artery.
  • the thread was inserted 9 ⁇ 1.0 mm into the internal carotid artery up to the middle cerebral artery.
  • a laser Doppler perfusion monitor (Perimed AB, JSrfalla, Sweden) was adhered to the right temporal aspect of the animal's skull and used to confirm middle cerebral artery flow disruption.
  • Control animals underwent sham surgery consisting of only anesthesia and carotid artery dissection. All animals were sacrificed after surgery. The animal's brain was removed, and the cerebral hemisphere was retained for either evaluation of infract volume and neuroscore or Western blot analysis. Determination of Infarct Volume
  • NASH imageJ 1.32j
  • mice were assigned a neurobehavioral score at 2 and 24 h following MCAO.
  • a previously reported scale was used (Stavrovskaya et al., 2004): 0, no neurological deficits; 1, failure to extend the left forepaw; 2, circling to contralateral side; 3, loss of ability to walk or righting reflex; and 4, death before 24 h.
  • cytochrome c Fractionation of cell and tissue. Mouse brain cytosolic fractionations were performed as described (Wang et al., 2003; Zhang et al., 2003b; Zhu et al., 2002). Released cytochrome c was analyzed by Western blot. Antibody to cytochrome c was purchased from PharMingen (San Diego, CA).
  • cytochrome c release To search for inhibitors of cytochrome c release, we developed a cell-free screening assay to identify drugs that would inhibit cytochrome c release from purified mouse liver mitochondria. Mitochondria were preincubated with the drug. To induce cytochrome c release, calcium was then added. Following a 30 minute incubation at 37°C, the mitochondria were pelleted. Using an ELISA, we quantified cytochrome c released from the mitochondria. We screened a library of 1 ,040 compounds for the ability to inhibit the release of cytochrome c from purified mitochondria of rat liver. The 1 ,040 compounds assembled by the Neurodegeneration Drug Screening Consortium of the NINDS provide a wealth of potentially neuroprotective agents. Most of them are FDA-approved drugs in current clinical usage (Heemskerk et al., 2002).
  • Fig. Ia is a representative graph generated from one of the 13 kits. The data for each set of 80 compounds is ultimately represented as a distribution of their impact upon cytochrome c release. Most drugs have little effect on the release of cytochrome c in response to Ca 2+ stimulation. However, there are outlying points, indicative of drugs that substantially stimulate or, more significantly, inhibit cytochrome c release. In Fig.
  • cytochrome c release To choose potential effective inhibitors of cytochrome c release, we selected drugs that inhibited release with a potency 1.5 standard deviations to the left of the mean (dotted line). In the primary screen, 48 compounds inhibited cytochrome c release below this value. Upon a secondary and confirmatory screen, 39 of these top candidates were confirmed to be effective at concentrations of 10 and 20 ⁇ M. We then selected among them on the basis of their known ability to cross the blood-brain barrier and their current usage (to treat other conditions) in humans. We rejected drugs with significant side effects and an inability to cross the blood-brain barrier, leaving 21 compounds (Fig. Ic). Thus, our cell-free assay identified just under 2% of the 1,040 compounds holding the greatest promise for use as neuroprotective agents.
  • Screen of cultured cells provides a class of inhibitors of cytochrome c release as potential drugs for neurodegeneration.
  • group II eight additional compounds might exhibit high potential for protection against neurodegeneration (Fig. 2b).
  • group II compounds ranked by IC50 at micromolar range from best to worst, are parthenolide (1.8 ⁇ M), probucol (8.3 ⁇ M), methazolamide (9.8 ⁇ M), hydroxy-progesterone caproate (11.7 ⁇ M), doxycycline hydrochloride (13.4 ⁇ M), melatonin (14.8 ⁇ M), minocycline hydrochloride (15.5 ⁇ M), and N-acetyl-DL-tryptophan (33.1 ⁇ M). Mephenytoin, one member of group II, might have a low potential for neuroprotectionn since its maximum protection value is low (23.3%).
  • Doxycycline hydrochloride was previously found to be neuroprotective (Yrjanheikki et al., 1998). Methazolamide and melatonin (N-acetyl-5-methoxytryptamine) have been reported to prevent SH-SY5Y neuroblastoma cell death following oxidative damage (Sarang et al., 2002).
  • minocycline is protective against neurodegeneration; it reduces tissue damage following ischemia and traumatic brain injury delays onset and extends survival in animal models of ALS and HD, and exhibits remarkable neuroprotective properties in experimental models of Parkinson disease and multiple sclerosis (Brundula et al., 2002; Chen et al., 2000; Friedlander, 2003; Wang et al., 2003; Wu et al., 2002; Yrjanheikki et al., 1998; Zhu et al., 2002).
  • Methazolamide and melatonin offer neuroprotection in cellular model of ischemia in vitro.
  • Methazolamide is currently used as a diuretic in humans, having been approved by the FDA as a treatment for glaucoma. At the molecular level, it is a noncompetitive inhibitor of carbonic anhydrase (Lindskog, 1997). However, there are no reports of methazolamide as a potential neuroprotective drug for neurodegenerative diseases. Like methazolamide, melatonin, a pineal-gland hormone implicated in the control of circadian rhythms, efficiently blocks cytochrome c release from purified mitochondria and crosses the blood-brain barrier. Melatonin is currently administered to blind patients, who tolerate high doses without substantial side effects.
  • melatonin is known to scavenge free radicals, thereby acting indirectly as an anti-oxidant, it is being investigated as a therapy for ALS, HD, Parkinson disease, and damage from stroke (Reiter et al., 1999; Reiter et al., 2003; Sarang et al., 2002).
  • methazolamide and melatonin are neuroprotective in mutant-htt ST14A striatal cells under a non-permissive temperature shift (Fig. 2b).
  • test drugs to block cell death is associated with the ability to protect against loss of ⁇ ⁇ m in living cells.
  • mitochondrial dysfunction with the upstream loss of ⁇ m is the key activating event underlying neuronal cell death in neurodegeneration (Friedlander, 2003; Wang et al., 2003).
  • the dissipation of the ⁇ m is a physiologic change accompanying programmed cell death (Hengartner, 2000).
  • As an upstream event of PT induction the dissipation of ⁇ m, causing the permeability transition pore in mitochondrial membranes to open, has been implicated as a critical effector of cell death (Tatton and Chalmers-Redman, 1998). Electrostatically charged mitochondria can be visualized in biological tissue by staining.
  • Rh 123 is such a paotentiometric dye already used to monitor mitochondrial function in apoptotic cells (Wang et al., 2003; Zhang et al., 1999).
  • minocycline acts, at least in part, by inhibiting release of cytochrome c and the dissipating the ⁇ m (Wang et al., 2003).
  • Methazolamide and melatonin are not inhibitors of the mitochondrial permeability transition (mPT) in isolated mitochondria.
  • the mitochondrial permeability transition represents a multiprotein complex composed of proteins of the mitochondrial matrix, including the outer and inner membranes (Zoratti et al., 2005).
  • Various pathologic factors such as high concentrations OfCa 2+ , pro-oxidants, thiol cross-linking reagents, and proapoptotic cytosolic proteins might induce activation of mPT and its irreversible opening.
  • mitochondrial dysfunction the loss of ⁇ m is an upstream step; activation of mPT is the next event.
  • the positive control minocycline can block both the loss of ⁇ m in neuronal living cells and activation of mPT on isolated mitochondria (Wang et al., 2003; Zhu et al., 2002), whereas methazolamide and melatonin blocked only the upstream step of mitochondrial dysfunction in neuronal living cells but not the next event of mPT activation in isolated mitochondria; we therefore suggest the mechanisms of action and inhibition of methazolamide are not identical to those of melatonin and minocycline.
  • Methazolamide like melatonin, decreases infarct size and neurologic impairment after MCAO.
  • Minocycline rescues neurons in cellular models of cell death and decreases the volume of the infarct from focal ischemia (Yrjanheikki et al., 1999). Since methazolamide and melatonin offer neuroprotection in cellular model of ischemia in vitro, we further tested whether they have the potential to ameliorate ischemic damage in vivo.
  • the infarct size in mice treated with methazolamide or melatonin was significant reduced as compared with that of DMSO-treated stroke control mice (Fig. 4a,b).
  • Caspase-mediated mitochondrial cell death pathways play important roles in neurodegenerative diseases (Beal, 1999; Chan, 2004; Friedlander, 2003; Wang et al., 2003; Zhang et al., 2003b; Zhu et al., 2002), and cytochrome c release and caspase-3 activation have been reported in mice with ischemic damage (Chan, 2004; Kim et al., 2000; Zhang et al., 2003b).
  • methazolamide and melatonin which inhibited cytochrome c release in isolated mitochondria, prevented cytochrome c release and caspase-3 activation.
  • methazolamide and melatonin two other cytochrome c release inhibitors, selected on the basis of their activity in the cell- free system and in cultured cells, are protective against cell death and ischemic stroke.
  • Our assay different with the large scale high through put, provides a rapid small scale drug screen focused on the FDA-approved bioactive compounds. Our study therefore provides a rapid assay for the rational design, screen, and discovery of drugs effective against neurodegeneration.
  • methazolamide and melatonin inhibited polyglutamine-induced cell death in a cellular model of HD, and in PCN, the primary targets of degeneration in HD are the striatal and cortical CNS neurons (Li, 1999; Reiner et al., 1988), these data should assist in the development of therapeutic strategies for chronic neurodegeneration of HD.
  • the FDA-approved drugs methazolamide and melatonin may thus be novel therapy for neurodegeneration.
  • melatonin is significantly effective in blocking striatal and PCN cell death through preventing the loss of ⁇ m and that it has a neuroprotective role in mice with ischemic stroke are complementary to the findings that melatonin protects against neurodegeneration (Feng et al., 2004; Jacob et al., 2002; Pei et al., 2003; Reiter et al., 1999; Tunez et al., 2004; Willis and Armstrong, 1999).
  • Methazolamide is a noncompetitive inhibitor of carbonic anhydrase.
  • Carbonic anhydrase is a zinc-containing enzyme that catalyzes the reversible hydration of carbon dioxide: CO 2 + H 2 ⁇ — >HCO3(-) + H + .
  • Drugs such as methazolamide and acetazolamide target carbonic anhydrase in the treatment of glaucoma (Lindskog, 1997). However, whether the neuroprotection provided by methazolamide involves carbonic anhydrase is not known.
  • calcimycin in group I there are four antibiotic compounds: calcimycin in group I, minocycline and doxycycline in group ⁇ , and metampicillin in group HI.
  • Two known neuroprotective agents, minocycline and doxycycline are both tetracycline-derivatives and have similar chemical structures (Fig. 5).
  • the relationship between the chemical structure of the compounds and their functionality is assessed.
  • Structural analysis showed that three group I compounds, fendiline hydrochloride, ritanserin, bepridil hydrochloride, and one group IJJ compound, meclizine hydrochloride, have a commonpharmacophore, with two phenyl rings and one positively charged center (nitrogen) (Fig. 5).
  • the drugs were obtained from the 1,040 compounds National Institute of Neurological Disorders and Stroke library by Custom Collection of Microsource Discovery Systems Inc., or were obtained from Sigma.
  • Mouse liver mitochondria were purified following the previously described method 7 .
  • An aliquot of 100 ⁇ l of (0.1 mg/ml) mouse liver mitochondrial preparation was preincubated with compounds from the NINDS drug library at a final concentration of 20 ⁇ M (diluted with assay medium by 500x from the 10 mM stock solution in DMSO) for 5 min in MRM buffer (250 mM sucrose, 1OmM Hepes, pH 7.5, ImM ATP, 5 mM sodium succinate, 0.08 mM ADP, 2 mM K 2 HPO 4 , pH7.5) at a concentration of 0.5 mg ml "1 .
  • MRM buffer 250 mM sucrose, 1OmM Hepes, pH 7.5, ImM ATP, 5 mM sodium succinate, 0.08 mM ADP, 2 mM K 2 HPO 4 , pH7.5
  • Mitochondria were then incubated with 20 ⁇ M CaCl 2 at 30 0 C for 30 min (CaCl 2 ). Mixtures were centrifuged at 10,000 x g at 4°C for 10 min and the supernatant evaluated using a Cytochrome c ELISA kit (R&D systems).
  • Enzyme-linked immunoabsorbent assay The NINDS library was obtained as 13 kits with 80 compounds in each. A single 96-well ELISA plate was dedicated to each kit of 80 compounds. Drugs were assayed with a separate mitochondrial preparation for each kit. For each sample within a given 96-well plate, the absorbance at 450 nm (a measure of cytochrome c concentration) was corrected by subtracting the background reading at 540 nm. Cytochrome c concentration was calculated by interpolating these values on a standard curve constructed for each plate.
  • the cell line ST14A were striatal cells conditionally immortalized by transfection with a temperature-sensitive form of the SV40 large T-antigen.
  • ST14A cells stably expressing a mutant huntingtin fragment (N548mu [ntl955— 128], mutant-htt ST14A) cells were cultured as described .
  • Mutant-htt ST14A cells were kindly provided by Dr. Maria Cattaneo (University of Milan, Italy). Cells were shifted to the non-permissive temperature of 37°C in serum-deprived medium (SDM) for the indicated times 6 . All experiments used 2 hours of pre-incubation with test drugs.
  • SDM serum-deprived medium
  • PCNs primary cerebrocortical neurons
  • PCNs were incubated with 1 mM H 2 O 2 or 500 ⁇ M NMDA for 18-20 hours, or OGD as described previously 5 .
  • the culture medium was replaced with glucose-free Earle's balanced salt solution with or without different concentrations of test drugs, and the cells were placed in an anaerobic chamber with BBL GasPak Plus (Becton Dickinson), which reduces the oxygen concentration to DlOO ppm within 90 min.
  • Control cells were incubated in Earle's balanced salt solution with glucose in a normoxic incubator for the same period. After 3 hours, OGD was terminated by a return to normal culture conditions. Cell death was determined by MTS or LDH assay.
  • MTS assay MTS assay. The assay was performed according to the manufacturer's instructions (Promega). Cell viability was assessed on mutant-htt STl 4 A cells using the 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTS) assay, a measure of mitochondrial function and thus cell survival.
  • MTS 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide
  • Lactate dehydrogenase (LDH) assay was performed according to the manufacturer's instructions (Roche Products). Briefly, reaction mixture (100 ⁇ l) was added to conditioned media (100 ⁇ l) removed from plates or dishes after centrifugation at 250 x g for 10 min. Absorbance of samples at 490 nm was measured in an ELISA reader after 15 min of incubation at room temperature. The same volume of medium "was used as the background control.
  • Mutant-htt STl 4A cells and mouse brain cytosolic -fractionations were performed as described 5"7 . Briefly, cells or brain samples were homogenized (1OmM HEPES, pH 7.4, 25OmM sucrose, 1OmM KCl, 1.5mM MgC12, ImM EDTA, ImM EGTA, ImM dithiothreitol (DTT), plus protease inhibitor cocktail. Released cytochrome c or Smac/Diablo was analyzed by Western blot. Antibody to cytochrome c was purchased from PharMingen. Smac/Diablo antibody from Novus Biologicals (Littleton, CO).
  • Mutant-htt ST14A cells were treated as indicated on chamber slides. The cells were incubated with 1.5 ⁇ M Mitotracker Red CMXRos (Molecular Probes) prepared in prewarmed culture media for 45 min, and fixed in 3.7% paraformaldehyde prepared in prewarmed culture media for 5 min, and then in ice-cold acetone for 5 min. Blocking was done in 5% BSA in PBS for 30 min. Cells then were incubated with antibodies to cytochrome c and incubated with FITC-conjugated secondary antibodies. Deconvoluted images were taken with a Nikon ECLIPSE TE 200 fluorescence microscope and processed by using IP LAB software (Spectra Services, Webster, NY).
  • Caspase Activity Assays Cell extracts and enzyme assays were performed with the Apo Alert caspase fluorescent assay kit as described 6 ' 53 and according to the manufacturer's instructions (Clontech). Briefly, 1 x 10 6 mutant htt ST14A cells were harvested in cell lysis buffer, and incubated on ice for 10 min. The cell lysates were centrifuged at 10,000 x g at 4°C for 3 min to precipitate cellular debris, and the supematants were evaluated for caspase activity by adding caspase substrate and 2x reaction buffer/DTT mixture.
  • Caspase-3-like substrate Ac-DEVD-AFC was from PharMingen, caspase-9-like substrate Ac-LEHD-AFC was from Calbiochem. Released AFC was quantified in a Bio-Rad Versa Fluoro Meter (excitation at 400 run and emission at 505 run).
  • mPT mitochondrial permeability transition
  • Mitochondria swelling was induced by Ca 2+ , tert-butylhydroperoxide (tBH), PhAsO, or diamide and monitored in standard spectroscopic assay at 540 nm. Methazolamide and melatonin were respectively applied to mitochondria. Mitochondrial physiological parameters including membrane potential ( ⁇ ), Ca 2+ uptake capacity, redox state of pyridine nucleotides and swelling was performed simultaneously on multichannel dye fluorimeter (C&L Instruments, Inc., www.fluorescence.com, Baranov et al., manuscript in preparation).
  • Mitochondrial membrane potential was estimated using TMRM (60 nM) (Molecular probes) fluorescence intensity changes at excitation and emission wavelengths 543 and 590 nm. Mitochondrial Ca 2+ uptake and release capacity was measured as changes of extramitochondrial [Ca 2+ ] followed by fluorescence intensity of CaGreen-5N (125 nM) (Molecular probes) at excitation and emission wavelengths 482 and 535 nm. Redox state of pyridine nucleotides was followed by measuring NADH autofluorescence at excitation and emission wavelengths 350 and 450 nm. Mitochondrial swelling was measured as a function of light scattering at ⁇ excitation and emission wavelengths 587 nm.
  • Focal cerebral ischemia was produced as described previously 5 . Briefly, anesthesia was induced in 6- to 8-week-old C57BL/6J mice (body weight 20-24 gr.) (Jackson Laboratory) with 2% (vol/vol) isoflurane (70% N 2 ⁇ /30% O 2 ) and was maintained with a 1-0.5% concentration. Rectal temperature was maintain ⁇ d between 37.0 and 37.5°C with a heating pad (Harvard Apparatus). Focal cerebral ischemia was induced by an intraluminal 7-0 nylon thread with a silicone tip (180 ⁇ m diameter) introduced into the right cervical internal carotid artery.
  • the thread was inserted 9 ⁇ 1.0 mm into the internal carotid artery up to the middle cerebral artery.
  • a laser Doppler perfusion monitor (Perimed AB) was adhered to the right temporal aspect of the animal's skull and used to confirm middle cerebral artery flow disruption.
  • Control animals underwent sham surgery consisting of only anesthesia and carotid artery dissection. All animals were sacrificed after surgery. The animal's brain was removed, and the cerebral hemisphere was retained for either evaluation of infract volume or Western blot analysis. All animal procedures were conducted in accordance with protocols approved by the Harvard Medical School Animal Care Committee.
  • MCAO Drug treatment in MCAO model.
  • Control animals were treated with a normal saline vehicle control containing 0.4% dimethyl-sulfoxide (DMSO-Sigma, St. Louis, MO, USA) and 3% Tween-20 (Sigma, St. Louis, MO, USA).
  • mice were assigned a neurobehavioral score at two and 24 hours following MCAO.
  • a previously reported scale was used 20 ' 54 : O 7 no neurological deficits; 1, failure to extend the left forepaw; 2, circling to contralateral side; 3, loss of ability to walk or righting reflex; and 4, death before 24 hours.
  • R6/2 mice (Jackson Laboratories, Bar Harbor, Maine) were randomly assigned to two groups. At 6 weeks of age, mice were treated with daily intraperitoneal injections of either saline or methazolamide (40 mg/kg or 20 mg/kg, Sigma, St. Louis, Missouri). For all experiments, each group had equal numbers of males and females. Muscle strength and coordination were evaluated weekly using a rotarod (Columbus Instruments). Disease onset was defined as the first day that the mouse failed to remain seven minutes at 15 rpm. Mortality was defined as the age at death or when the mouse was unable to right itself within 30 seconds. Body weight was recorded weekly.
  • Tween 20 Tween 20
  • Sections were then incubated overnight in TBS-T with 0.1% sodium azide and mouse antihuntingtin (1:500, Chemicon, Temecula, CA) and anti-DARP-32 (1:500, Santa Cruz Biotechnology, Santa Cruz, CA). Sections were rinsed in TBS-T, followed by a 2-3 hour incubation in secondary antibodies. Sections were then rinsed three times in TBS-T. Antibody complexes were visualized using diaminobenzidine. Preabsorbtion with excess target protein, or omission of either primary or secondary antibody, was used to demonstrate antibody specificity and background generated from the detection assay. Tissue sections were examined using a Nikon Eclipse E800 microscope with a Spot RT digital camera.
  • Stereology/Quantitation Serial-cut coronal tissue-sections from the most rostral segment of the neostriatum to the level of the anterior commissure (Interaural 5.34 mm/Bregma 1.54 mm to Interaural 3.7 mm/Bregma -0.10 mm), were used for huntingtin aggregate analysis. Unbiased stereological counts of huntingtin-positive aggregates (>1.0 ⁇ m) were obtained from the neostriatum in 6 mice each from methazolamide-treated and saline-treated R6/2 mice at 25 weeks using Neurolucida Stereo Investigator software (Microbrightfield, Colchester, VT).
  • the total areas of the rostral brain, ventricles, and neostriatum were defined in serial sections. Counting frames were randomly sampled within the neostriatum for huntintin aggregate counts. The optical dissector method was employed estimating the number of huntingtin-positive aggregates. Striatal neuron areas were analyzed by microscopic videocapture using a Windows-based image analysis system for area measurement (Optimas, Bioscan Incorporated, Edmonds, WA). The software automatically identifies and measures profiles. All computer identified cell profiles were manually verified as neurons and exported to Microsoft Excel. Experiments were in accordance with protocols approved by the Harvard Medical School Animal Care Committee.
  • cytochrome c release To search for inhibitors of cytochrome c release, we have developed a cell- free screening assay to identify drugs that inhibit cytochrome c release from mouse-liver mitochondria. Isolated mitochondria are preincubated with the test drug for five minutes. To induce cytochrome c release, the organelles are treated with 20 ⁇ M Ca 4+ for 30 minutes at 37°C. After removing the mitochondria by centrifugation, the supernatant is analyzed by an enzyme-linked immunosorbent assay (ELISA) to determine the concentration of cytochrome c. This procedure was used to test all 1,040 compounds in a library assembled by the Neurodegeneration Drug Screening Consortium of the NINDS.
  • ELISA enzyme-linked immunosorbent assay
  • Fig. Ia shows the distribution of activities among the 80 compounds compromising one of 13 "kits” that constitute the library. Equivalent data were gathered for the 12 other kits. To generate Fig. Ib, the data for each of these 13 sets of compounds are normalized so that the average in each "kit” is the same. The colored curves generated in this manner are then summed to form a grand distribution covering the entire NTNDS library (solid black line). Most drugs have little effect on the release of cytochrome c in response to Ca 2+ - stimulation. However, there are outlying points, indicative of drugs that substantially stimulate or, more significantly, inhibit cytochrome c release.
  • Inhibitors of cytochrome c release are protective of cultured cells
  • IC 5 0 for these compounds is particularly low, and they offer strong protection at the saturating concentration. They are fendiline hydrochloride, calcimycin, dipyrone, and bepridil hydrochloride, compounds with IC 50 of 7 nM, 7 nM, 15 nM, and 128 nM, respectively.
  • Ritanserin and azathioprine are unlikely to prove neuroprotective because they fail to provide even 30% protection when present at saturating concentrations. Nevertheless, their low IC 50 of 50 nM and 124 nM, respectively, relegate them to Group I (Fig. 2a).
  • Doxycycline hydrochloride has documented neuroprotective potency 16 .
  • Methazolamide and melatonin N-acetyl-5-methoxytryptamine
  • minocycline is protective against various acute and chronic afflictions of the CNS. It reduces tissue damage following ischemia and traumatic brain injury, delays onset and extends survival in animal models of ALS and HD, and exhibits remarkable neuroprotective properties in experimental models of Parkinson disease and multiple sclerosis 1>6>7 - 15 - 18 ' 30
  • Some reports fail to demonstrate protection using minocycline.
  • minocycline-mediated neuroprotection has been accomplished in a broad verity of models in a number of independent laboratories 31"33 .
  • the 16 drugs found to be protective of cultured cells three were selected for investigation in vivo.
  • Methazolamide and melatonin are neuroprotective of primary cerebrocortical neurons.
  • methazolamide was chosen because it is tolerated by patients and, too a significant extent, crosses the blood-brain barrier. It is currently used as a diuretic in humans, being approved by the FDA for the treatment of glaucoma. At the molecular level, it is a noncompetitive inhibitor of carbonic anhydrase. However, there are no reports of methazolamide being tested for its benefits to patients with neurodegenerative diseases.
  • the pineal-gland hormone melatonin a drug that is better studied than methazolamide, also holds potential for the treatment of chronic neurological diseases. Though it hasn't been tested in that capacity, melatonin is known to cross the blood-brain barrier.
  • melatonin is used to treat blind patients. Melatonin scavenges free radicals, making it a de facto anti -oxidant. This property of the compound motivated studies of its potential benefits to patients with ALS, HD, and Parkinson disease and to persons who have suffered a stroke. As described above, we found that both methazolamide and melatonin inhibit mitochondrial cytochrome c release and protect mutant htt ST14A cells from cell death upon temperature shift.
  • methazolamide is neuroprotective is the first in the literature. It is of interest to determine the compound's effects on the molecular physiology of the cell. The ability of melatonin to inhibit neuronal cell death is well known, but again its effects on subcellular processes are poorly understood. Investigation of mitochondrial cell death pathways, both caspase-dependent and independent, is a promising avenue of research, as they are of documented importance to neurodegeneration ⁇ 5"9 - 36 - 37 . I n particular, the release of cytochrome c and other apoptogenic factors from these organelles triggers sequential maturation of caspase-9 and caspase-3, as well as caspase-independent cell death events.
  • the mutant htt ST14A cell line is a useful system in which to pursue the above investigations. Because these conditionally-immortalized striatal cells carry a gene for temperature-sensitive large T antigen of SV40 and another one encoding polyglutamine-expanded huntingtin, they undergo cell death upon transfer to the nonpermissive temperature of 37 0 C 6>23 . Cytochrome c localization was evaluated by immunocytochemistry using a fluorescence microscope and deconvolution software. Cells were co-labeled with anticytochrome c (Fig. 6a, green in upper panels) and a mitochondrial marker, MitoTracker (Fig. 6a, red in middle panels). Merged images demonstrate localization of cytochrome c immunoreactivity with mitochondria (Fig.
  • ⁇ m Dissipation of the electrostatic potential gradient across the mitochondrial membrane ( ⁇ m ) is part of the mitochondrial dysfunction that triggers the caspase cascade. Consequently, the collapse of ⁇ m is a critical change underlying the physiological process of neuronal cell death and resulting neurological disease 1 ⁇ 12 - 39 . Presumably, the dissipation of ⁇ T m results from opening of the permeability-transition pore. Certainly, a membrane channel that is large enough to allow passage of apoptogenic factors would enable the rapid equilibration of protons and other charged particles. To follow the biochemical process, we observed the effects of test drugs on the localization of rhodamine 123 (Rh 123) within the cell.
  • Rh 123 is a fluorescent, hydrophobic, and positively charged molecule, and therefore one that accumulates within electrostatically charged mitochondria. This chemistry causes stained cells to exhibit a punctate pattern of fluorescence except once their mitochondria have become depolarized. Indeed, observation with a fluorescence microscope of cells stained with Rh 123 is commonly used to monitor mitochondrial function in cells undergoing apoptosis ' . As noted above, the neuroprotective properties of minocycline have been linked to the drug's effects on mitochondria, i.e., inhibition of cytochrome c release, compromised mitochondrial swelling, and preservation of ⁇ m 6>?
  • Methazolamide and melatonin are not inhibitors of the mitochondrial permeability transition (mPT) in isolated mitochondria.
  • the mitochondrial permeability transition pore consists of a multimeric complex of proteins spanning the inner and outer membranes 41 ' 42 . Its opening is a component of mitochondrial dysfunction, itself a result of such biochemical stresses as high concentrations of Ca 2+ ion, oxidizing agents, compounds that cross-link thiols, and proapoptotic cytosolic proteins.
  • the loss of ⁇ m is an early event in the cell death pathway and an important trigger of the caspase cascade.
  • Methazolamide was associated with a slightly increased sensitivity to Ca 2+ -overload, but this would be expected to, if anything, slightly facilitate cell death, not to be protective.
  • the data in Figure 7 C-F suggest that melatonin and methazolamide do not mediate their protective effects by acting upon basic mitochondrial physiology, including mitochondrial redox status and substrate, proton, electron, or Ca 2+ transport. These data are most consistent with effects mediated outside the inner mitochondrial membrane/mitochondrial matrix, including potential targets such as bcl-2 family members (e.g., bax) and the elements involved in release of pro-apoptotic proteins. Studies are in progress to address these potential mechanisms.
  • the data in Figure 7 C-F also suggest that these agents will likely be free of mitochondrial-based detrimental effects, enhancing their potential utility for clinical applications.
  • Methazolamide and melatonin decrease cerebral ischemia-induced injury.
  • minocycline decreases the volume of the infarct produced by focal ischemia 44 .
  • methazolamide and melatonin like minocycline, forestall cell death due to OGD, suggesting that they may be protective in cerebral ischemia.
  • pretreatment with methazolamide or melatonin caused mice to develop smaller infarcts following middle cerebral artery occlusion (MCAO) than do animals injected with the vehicle (Fig. 8a,b).
  • MCAO middle cerebral artery occlusion
  • their post-ischemic behavior was less severely impaired as judged by the criteria of Hara et. al. AS (Fig. 8a,c).
  • cytochrome c release and caspase-3 activation are processes important to a broad spectrum of acute and chronic neurodegenerative diseases 1 ' 5"9 .
  • cytochrome c release and caspase-3 activation are observed in brains of mice that have recently suffered ischemic damage 5 " 8 ' 48 .
  • methazolamide and melatonin diminish cytochrome c release and caspase-3 activation as stimulated by such acute insult.
  • methazolamide and melatonin reduced cytochrome c release and caspase-3 activation in ischemic tissue (Fig. 8d,e).
  • dipyrone a Group I drug as defined in Fig. 2, protects primary cerebral cortical cells that are challenged with OGD.
  • dipyrone was found to be the drug that best protects mu htt STl 4A cells from cell death upon temperature shift (up to 70 % inhibition of cell death, IC 5 0 - 15 nM).
  • dipyrone Similar to methazolamide and melatonin, dipyrone also inhibited the loss of ⁇ m as induced by OGD (Fig. 7b). In addition, MCAO produced smaller infarcts and caused less severe behavioral changes in mice treated with dipyrone than in those injected with the saline vehicle (Fig. 9c,d).
  • Methazolamide delays disease onset and mortality in R6/2 mice, an animal model of chronic neurodegeneration
  • methazolamide In light of its ability to counter cell death of PCNs and striatal cells, methazolamide is apt to benefit test animals suffering from chronic neurodegenerative diseases. As noted above, we have shown that methazolamide decreases injury following cerebral ischemia (Fig. 8). This property is reminiscent of minocycline's ability to rescue cultured neurons, decrease the impact of ischemic stroke, and slow neurodegeneration in R6/2 mice 6 ' 17 " 33 . Motivated by these findings in animals that suffered acute injury, we proceeded to investigate methazolamide's ability to slow chronic neurodegeneration. R6/2 mice were chosen as the animal model because they suffer neurodegeneration reminiscent of that in FDD.
  • R6/2 mice are widely used for studying novel drug therapies for the human disease 17*33 ' 51 ' 52 .
  • R6/2 mice received daily intraperitoneal .injections of 20 mg/kg or 40 mg/kg methazolamide.
  • Control R6/2 mice were injected with saline vehicle. Body weight was monitored weekly. Both the test and control groups were comprised of equal numbers of males and females.
  • methazolamide-treatment significantly delayed disease onset in R6/2 mice by 15% and 27% in the 20mg/kg and 40 mg/kg groups respectively when compared to saline-treated controls (Fig. lOa-c).
  • methazolamide treatment significantly extended their lifespan by 11% (20 mg/kg) and 20% (40 mg/kg) (Fig. 1Od).
  • the time course of weight loss was not significantly altered (Fig. 10c). Marked neuroprotection at the tissue level was observed in methazolamide-treated R6/2 mice, as evidenced by reduced brain weight loss (Fig- 1Oe).
  • methazolamide administration also significantly ameliorated the striatal neuronal atrophy present in the saline-treated R6/2 mice (WT littermate control: 136.8 ⁇ 9.8 gm 2 ; methazolamide-treated R6/2 mice: 105.7 ⁇ 15.7 gm 2 ; saline-treated R6/2 mice: 57.4 ⁇ 21.6 gm 2 ; (F( 2 ,i 8 ) 21.61, *p ⁇ 0.01) (Fig. 1Of).
  • DARP32-immunopositive neurons in medium-sized striatal neurons, we performed an immunohistochemical analysis of DARP32-immunopositive neurons in the R6/2 mice, hi comparison to the saline-treated R6/2 mice, methazolamide markedly reduced the loss of DARP32 immunoreactive striatal neurons.
  • cytochrome c i.e., that which remains in the supernatant once the organelles have been removed by centrifugation, can easily be detected by an enzyme-linked immunosorbent assay (ELISA).
  • ELISA enzyme-linked immunosorbent assay
  • Those compounds that reduce the resulting signal are promising candidates for neuroprotective drugs.
  • Compounds from the resulting "short list" are then assayed for their ability to rescue cultured cells challenged with a cell death stimuli, e.g., OGD or incubation at the nonpermissive temperature. Species that are protective in these cellular systems are further evaluated in trials on animal models.
  • biologically safe and potentially curative agents can be tested on human patients afflicted by both acute neurological injury and chronic neurodegenerative.
  • Minocycline and doxycycline two compounds known a priori to slow neurodegeneration 1,6,7,15 - 18 ⁇ ⁇ 6 particularly effective at inhibiting the release of cytochrome c from purified mitochondria. These observations are reassuring, as they suggest that the in vitro assay has predictive value.
  • Li, L.Y., Luo, X. & Wang, X. Endonuclease G is an apoptotic DNase when released from mitochondria. Nature 412, 95-9. (2001).

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Abstract

L'invention concerne des produits et des méthodes pour inhiber la libération de cytochrome C et/ou la mort cellulaire. Les méthodes de l'invention permettent de prévenir et de traiter des maladies associées à la libération de cytochrome C et/ou des troubles tels que : maladie d'Alzheimer, maladie de Parkinson, maladie de Huntington, SLA, épilepsie, accident cérébrovasculaire, trauma crânien, lésion de la mœlle épinière, lésion hypoxique périnatale, dégénérescence rétinienne, ischémie cérébrale ou d'autre organe terminal induisant une mort cellulaire associée à une chirurgie neurale, vasculaire, cardiaque ou de transplantation, infarctus du myocarde, neuroprotection périopératoire pendant endartérectomie carotidienne, stents carotidiens et cardiaques, réparation d'anévrisme aortique abdominal (AAA) ou diabète.
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US5827819A (en) * 1990-11-01 1998-10-27 Oregon Health Sciences University Covalent polar lipid conjugates with neurologically active compounds for targeting
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US5681711A (en) * 1995-03-27 1997-10-28 University Of California-Los Angeles Method for promoting apoptosis in mammalian neual cells
US20040072205A1 (en) * 2002-05-01 2004-04-15 Friedlander Robert M. Methods for treating neuronal disorders
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US10471075B1 (en) 2015-04-30 2019-11-12 Amag Pharmaceuticals, Inc. Methods of reducing risk of preterm birth
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