WO2007059039A1 - Thyroxine derivatives for preconditioning against stroke - Google Patents

Abstract

Methods of preconditioning against injury due to ischemic and/or hypoxic events by administering a thyronamine derivative are provided.

Description

THYROXINE DERIVATIVES FOR PRECONDITIONING AGAINST STROKE

CROSS REFERENCE TO RELATED APPLICATION

[001] This application claims benefit of the prior filing date of U.S. provisional patent application 60/735,733, filed November 11, 2005, the disclosure of which is incorporated herein in its entirety.

ACKNOWLEDGMENT OF GOVERNMENT SUPPORT '

[002] Aspects of this invention were made with United States government support pursuant to grant no. POI NS035965 from the National Institute of Neurological Disorders and Stroke (NINDS). The United States government may have certain rights in the invention.

FIELD

[003] This disclosure relates to the field of neuroprotection. More specifically, the present disclosure relates to administration of a thyroxine derivative to inhibit cellular and organ damage due to excitotoxic injury, ischemia and/or hypoxia.

BACKGROUND

[004] Thyroxine (T4) is the principal secreted form of thyroid hormone and makes up 95% of all thyroid hormone found in the circulation (Mortoglou et ah, Hormones, 3: 120-126, 2004). T4 is deiodinated in target tissue to 3,5,3'-triiodothyronamine (T3), the more active form of thyroid hormone, which has a higher affinity for thyroid hormone receptors found on the nuclear membrane (Hiroi et ah, Proceeding of the National Academy of Science, 103 : 14104- 14109, 2006). Both T4 and T3 regulate metabolism and tissue thermogenesis by gene transcription, a process that takes hours to come into effect (Scanlan et ah, Nat. Med., 10:638- 642, 2004). Physiological effects of thyroid hormone include the induction of hyperthermia and increased cardiac output. Thyroid hormone can be further deiodinated and decarboxylated into 3-iodothyronamine (TlAM) and thyronamine (TOAM) (Scanlan et ah, Nat. Med., 10:638- 642, 2004). These newly characterized thyronamines have been found in rodent brain, peripheral organs and blood. TlAM and TOAM have the opposite effect to T4 and T3, rapidly inducing hypothermia in a mechanism independent of gene transcription. TlAM and TOAM are not ligands for traditional thyroid hormone receptors but are agonists for trace amine associated receptor 1 (TAARl), a G-protein coupled receptor found on the plasma membrane (Scanlan et ah, Nat. Med., 10:638-642, 2004). [005] Intraperitoneal injection (ip) of TlAM and TOAM induces hypothermia within 30 minutes, with no long-term adverse effects. This hypothermic response occurs in the absence of shivering and piloerection, indicating that the hypothermic effect of TlAM and TOAM is not opposed by the body's natural homeothermic response.

[006] The induction of hypothermia is a profoundly neuroprotective treatment for stroke (Kammersgaard et al, Stroke, 31:2251-2256, 2000; Schwab et al, Acta. Neurochir. Suppl, 71 : 131-134, 1998; Schwab et al., Nervenarzt., 70:539-546, 1999; Schwab et al, Stroke, 29:2461-2466, 1998; Colbourne and Corbett, J. Neuroscl, 15:7250-7260, 1995; Colbourne et al., J. Cereb. Blood Flow Metab., 20: 1702-1708, 2000; Colbourne et al., MoI. Neurobiol, 14:171-201, 1997; Colbourne and Corbett, Brain Res., 654:265-272, 1994; Nurse and Corbett, J. Cereb. Blood Flow Metab., 16:474-480, 1996; Yunoki et al, J. Neurosurg., 97:650-657, 2002; Krieger et al, Stroke, 32: 1847-1854, 2001). The protective effect is dependent on the duration and depth of hypothermia and its timing relative to the onset of stroke. It has not been possible to identify a single action of hypothermia that is responsible for its marked neuroprotective actions. It may work so well simply because it works through a multitude of protective mechanisms simultaneously, something that distinguishes it from most neuroprotective drugs. Mechanisms by which hypothermia confers benefit include improving the ratio between metabolic demand and substrate supply by reducing metabolic rate, reducing free radical formation, blunting reperfusion injury, and reducing the release of glutamate (Krieg AM., Annu. Rev. Immunol, 20:709-760, 2002). Hypothermia also has negative side effects, for example it can cause an inhibition of coagulation, cardiac arrythmia, pulmonary edema and an increased risk of infection due to immunosuppression. However these side effects can be effectively managed with primary care without causing long term adverse effects (Polderman KH, Intensive Care Med., 30:757-769, 2004).

[007] Current methods of achieving hypothermia force temperature below the internal homeostatic set point of a subject. Such methods include using anesthesia and packing ice around the whole body or the head, spraying alcohol or cold water directly onto the skin, and using intravascular cooling devices (Hammer and Krieger, Neurologist, 9:280-289, 2003). Mammals respond to such a forced reduction in temperature by shivering, increasing brown fat thermogenesis and vasoconstricting blood vessels. This homeostatic response can be prevented pharmacologically, but because pharmacokinetics changes according to temperature, ascertaining correct dosage is complex. [008] An alternative to inducing hypothermia by physical means is to use drugs (cryogens) to lower body temperature. Studies that have looked at the effect of administering hypothermia show that the sooner hypothermia is initiated after ischemia the greater the level of protection conferred (Maier et ah, J. Neurosurg., 94:90-96, 2001). The cannabinoid HU- 120 has been used to induce hypothermia and protect against ischemia in rats (Leker et ah, Stroke, 34:2000-2006, 2003). However, at doses that induce hypothermia, HU-120 has toxic side effects that limit its application for humans. Additional studies with hypothermia inducing cannabinoids have been more encouraging. The cannabinoid WIN 55,212-2 can induce therapeutic hypothermia with fewer side effects than HU-120, however WIN 55,212-2 has the limitation of requiring continuous intravenous infusion for the hypothermic effect to be maintained (Bonfils et ah, Neurochem. Int., 49(5):508-518, 2006). Another recent study demonstrates that hydrogen sulfide can be used to reduce temperature in rodents (Blackstone et ah, Science, 308:518, 2005). Yet hydrogen sulfide is also a mediator of cerebral ischemic damage which could counterbalance the beneficial effects of hypothermia if hydrogen sulfide were to be used as a treatment for stroke (Qu et ah, Stroke, 37:889-893, 2006).

[009] Thyroxine derivatives, such as TlAM and TOAM, have also been proposed as cryogens for the treatment of an already evolving stroke (U.S. Patent No. 6,979,750; Scanlan et ah, Nature Med., 10:638-642, 2004). These metabolites are naturally found in blood, brain and peripheral organs. Both TlAM and TOAM can be used as acute neuroprotective agents following the onset of the stroke. TlAM and TOAM induce an equivalent level of hypothermia following stroke, and when this effect is blocked neuroprotection is lost. This implies that the protective effect of acute administration of TlAM and TOAM is due to hypothennia.

SUMMARY

[010] It has now unexpectedly been found that thyronamine derivatives are useful as cytoprotective agents, for example in the prophylaxis of cellular damage induced by a variety of cellular insults. This disclosure concerns preconditioning agents that mediate cellular protection (such as neuroprotection during hypoxia), and provides methods for reducing and/or ameliorating damage due to excitotoxic, ischemic and/or hypoxic events, such as stroke and traumatic brain injury. Certain thyroxine derivatives, designated herein as thyronamine preconditioning agents, are administered prior to a potential incident of cellular injury, such as an excitotoxic, hypoxic and/or ischemic event. Following injection of the agent, mice show a pronounced drop in temperature (from 37°C to 31°C) that persists for several hours. Animals treated with thyronamine preconditioning agents exhibit reduced cellular damage due to ischemia as compared to control mice, or mice treated with other thyroxine derivatives. This finding allows these agents to be used in situations in which a likelihood of cellular injury (such as that caused by a hypoxic event such as ischemia) is increased, for example by administration to subjects who are at risk for neurological injury such as a stroke or who are about to undergo a surgical procedure or other activity in which such injury may occur. In particular examples, the preconditioning agent is administered at least 10 to 12 hours before the anticipated cellular injury (such as an ischemic event), or at least 24 to 48 hours prior to the event. In another example the agent is administered on a regular regimen to a subject at risk of cellular (such as neurological) injury, for example by administration of an effective protective amount to a subject at least once a week. In certain examples, the agent is chronically administered to a subject who has a neurodegenerative disease (such as Alzheimer's disease) that benefits from the cytoprotective effect of the agent. In other examples the agent is used to protect the brain against injury induced by repeated seizures (as in epilepsy).

[Oi l] In particular embodiments, the disclosed method is believed to provide the advantage of substantially avoiding neurological damage induced by injuries such as excititoxic injury or hypoxia (including ischemia), instead of merely attempting to limit the damage after it has already occurred.

[012] The foregoing and other objects, features, and advantages of the invention will become more apparent from the following detailed description, which proceeds with reference to the accompanying figures.

BRIEF DESCRIPTION OF THE DRAWINGS

[013] FIG. IA is a schematic illustration of TOAM and TlAM.

[014] FIG. IB is an illustration of 3-Methylthyronamine.

[015] FIG. 1C is an illustration of N-Methyl-O-(p-trifluoromethyl)benzyl-tyramine Hydrochloride, wherein X is H, Ri is CF₃, and R₂ is methyl.

[016] FIG. ID is an illustration of 3-iodotyramine hydrochloride. OPhenyl-3-iodotyramine hydrochloride: R] is H, R₂ is H; (9-(p-Fluoro)phenyl-3-iodotyramine hydrochloride: Ri is F, R₂ is methyl. [017] FIGS. 2A and B are graphs demonstrating that acute administration of TlAM and TOAM causes hypothermia and protection against ischemic injury. A) Temperature reduction following administration of TlAM or TOAM post stroke. Mice were i.p injected with vehicle or 50mg/kg of TlAM or TOAM 1 hour post reperfusion. Temperature was measured every hour for 6 hours post stroke and immediately before sacrifice (24 hours post stroke). In two separate groups of animals (Tl hypothermia blocked and TO hypothermia blocked) temperature reduction following TlAM and TOAM injection was prevented by maintaining animals on an adjustable heating pad. B) Infarct volume following TlAM, TOAM, TlAM hypothermia blocked and TOAM hypothermia blocked treatment following MCAO. N = 8 per group. Error bars represent standard deviation. * p value = <0.05 compared to vehicle injected.

[018] FIGS. 3A and B are graphs demonstrating preconditioning with TlAM and TOAM. Preconditioning with TlAM but not TOAM induces delayed tolerance to ischemic injury. A) Temperature reduction post injection with TlAM and TOAM. Mice were injected with 50mg/kg of TlAM or TOAM 2 days prior to MCAO. Temperature was measured for 8 hours post injection. In a separate group injected with 50mg/kg TlAM, mice were prevented from becoming hypothermic by maintaining them on an adjustable heating pad and maintaining their temperature as close to that of vehicle injected mice as possible (TlAM hypothermia blocked). B) Infarct volume following TlAM and TOAM preconditioning treatment and TlAM hypothermia blocked preconditioning treatment. N = 8 each group. Error bars represent standard deviation. * p value = <0.05 compared to vehicle injected.

[019] FIG. 4A is a graph illustrating TlAM and TOAM acute administration in vitro. Experiments were performed on mouse primary neuronal cultures. Cells were treated with the indicated doses of TlAM or TOAM immediately after OGD. Cell viability assays were performed 24 hours post OGD (N=3). Treatment with 5nM OPN peptide (EMD Biosciences) was used as a positive control. * p value = <0.05 compared to OGD control.

[020] FIG. 4B is a graph showing TlAM and TOAM preconditioning in vitro. Experiments performed on mouse primary neuronal cultures treated with the indicated doses of TlAM and TOAM 2 days prior to OGD. Cell viability assays were performed 24 hours post OGD (N=3). DETAILED DESCRIPTION Introduction

[021] This disclosure provides compositions and methods for preconditioning to protect against damage due to excitotoxic, ischemic and/or hypoxic events, such as those that occur with high frequency during many types of surgery, and are associated with traumatic brain injury, epilepsy and certain neural degenerative disorders, including Alzheimer's disease; The methods and compositions are therefore useful in the treatment of neurological conditions, such as those caused by hypoxia (including ischemia) and certain types of neurological disease (such as those in which hypoxia or excitotoxic injury are pathophysiological features).

[022] Hypothermia administered during ischemic events reduces global ischemic injury and provides lasting behavioral and histological protection in both young and old animals and remains the gold standard for studies of neuroprotection (Thornhill and Corbett, Canadian Journal of Physiology and Pharmacology, 79:254-261, 2001). Since cooling during ischemia is usually not possible, much research has been conducted to determine whether post-ischemic hypothermia is also beneficial. Early studies suggested that post-ischemic hypothennia was ineffective, but this was due to only brief durations of cooling being applied (2-3 hrs) (Dietrich et al., Journal of Cerebral Blood Flow and Metabolism, 13:541-549, 1993). Subsequent preclinical tests with greater durations of hypothermia of 6 hours or more have demonstrated that post-ischemic hypothermia is effective and that the therapeutic window expands as the duration of hypothermia is increased. For example with cooling periods of 48 hours significant long term protection of CAl hippocampal neurons can be achieved in the gerbil global ischemia model even when cooling is delayed by 12 hours (Colbourne et al., J. Neurosci., 19:4200-4210, 1999).

[023] Hypothermia can also be used as a preconditioning stimulus to prevent or reduce the extent of damage resulting from excitotoxic, ischemic and/or hypoxic events. Hypothermia induced tolerance commences within 6 hours, peaks at approximately 1 or 2 days and returns to baseline by about 7 days after preconditioning stimulation (Nishio et al., Ann N Y Acad. ScL, 890:26-41, 1999). Hypothermia induced tolerance is dependent on de novo protein synthesis and may be dependent on modification of proteins by SUMOylation (Nishio et al., Ann. NY Acad. Sci., 890:26-41, 1999; Lee et al, Journal of Cerebral Blood Flow & Metabolism, September 6, 2006). Thus, induction of hypothermia prior to excitotoxic, ischemic and/or hypoxic events is a valuable strategy for limiting cerebral injury during events or conduct that places a subject at high risk of an excitotoxic, ischemic and/or hypoxic event. [024] From a practical standpoint, hypothermia is inconvenient to induce as a preconditioning stimulus. Thus, to fully realize the potential of hypothermia for preconditioning against injury due to excitotoxic, ischemic and/or hypoxic events, it is necessaiy to identify cryogens that can be administered to subjects with minimal toxicity, and in a convenient form and dosage regimen. This disclosure provides for the first time pharmaceutical compositions that include a cryogen which can be administered as a preconditioning agent to protect a subject against excitotoxic, ischemic and/or hypoxic injury.

[025] Certain embodiments of the disclosed methods involve methods of preventing cellular damage due to excitotoxic, ischemic and/or hypoxic events by administering a class of thyroxine derivatives, designated herein as thyronamine preconditioning agents, prior to the onset of an event that produces an excitotoxic, ischemic and/or hypoxic condition or state. An example of such a state is a neurological injury, such as a neurological injury induced by trauma or disease in which hypoxia (including ischemia) or exicitoxic injury induces cellular damage. Accordingly, a first aspect of the disclosure relates to a method of protecting a cell in a subject (such as a human subject) against such injury. The method involves administering to the subject a therapeutically effective amount of a composition that includes the thyronamine preconditioning agent. The composition is administered prior to an event that produces or is likely to produce the neurological injury. Administration of the thyronamine preconditioning agent elicits a response by the subject that protects against cellular damage.

[026] Typically, the composition is administered to a subject selected on the basis of an increased risk for the neurological insult. In certain embodiments, the subject is selected prior to undergoing a surgical procedure that increases the risk of the adverse event, such as a vascular surgical procedure, for example, endarterectomy (such as carotid endarterectomy), pulmonary bypass or coronary artery bypass graft surgery. In other instances the subject is selected on the basis of participation in a contact sport or in combat, or in other activities that increase the risk of traumatic brain injury, which is associated with increased risk of excitotoxic, ischemic and/or hypoxic injury.

[027] Typically, the thyronamine preconditioning agent is administered at least about 6 or 10 hours prior to the exctitoxic, ischemic and/or hypoxic event, for example 6-12 or 10-12 hours before the event. In other examples, the agent is administered 24-48 hours prior to the event. For some applications, especially in subjects with a chronically increased risk of such a deleterious event (such as prolonged exposure to potential head trauma or a chronic neurological disease such as a degenerative neurological disease, such as Alzheimer's disease), multiple doses of the preconditioning agent are administered in sufficient proximity to one another to provide sustained neuroprotection. Typically, the thyronamine preconditioning agent is administered on a schedule calculated to maintain the preconditioning effect, such as at intervals of less than about 1 week, such as a biweekly interval, or a daily interval. The repeated doses can be maintained, such that the ultimate dose is delivered within 1 week prior to an anticipated episode of potential neurological injury (such as an excitotoxic, ischemic and/or hypoxic event). For example, a subject at increased risk of stroke (selected based on one or more medical indicators or criteria associated with increased risk of stroke, such as high blood pressure, cardiovascular disease, carotid atherosclerosis, prior ischemic event, etc.) can be administered a weekly or more often than weekly dose of the thyronamine preconditioning agent to induce and maintain a protected state. Similarly, subjects with chronic conditions that can result in neurological injury such as excitotoxic injury (for example subjects with epilepsy or Alzheimer's disease) can be administered repeated doses of a thyronamine preconditioning agent to protect against excitotoxic injury.

[028] Favorably, the cytoprotective method disclosed herein is suitable for preconditioning to protect neural cells, as well as muscle cells, liver cells, kidney cells, endothelial cells and immune system cells. For example, a thyronamine preconditioning agent can be administered to a subject prior to an excitotoxic, ischemic and/or hypoxic event (such as a stroke) to protect brain cells, such as hippocampal, cortical and/or other neurons. Similarly, a thyronamine preconditioning agent can be administered to a subject to protect muscle cells, such as cardiac muscle cells. In some embodiments, the thyronamine preconditioning agent is administered to a subject that engages in activities with an increased likelihood of traumatic brain injury. In some embodiments, the thyronamine preconditioning agent is administered to a pregnant woman to protect her fetus against hypoxia in utero. Such fetal protection can be of particular importance in events that can lead to fetal injury, such as perinatal hypoxia, placental insufficiency or a pregnancy associated hypertension (for example preeclampsia or eclampsia).

[029] Administration of the thyronamine preconditioning agent can be performed by any of a variety of routes, including oral, nasal and rectal routes as well as parenteral routes, such as intraperitoneal, intravenous, intramuscular, subcutaneous, subdermal and transdermal routes. In some applications, the composition can be administered by an intrathecal, intraventricular, and/or epidural route. Any thyronamine derivative and/or analogs thereof that induces a sufficient level and duration of hypothermia, and exhibits desirable toxicology characteristics can be administered as a thyronamine preconditioning agent. Specific examples of thyronamine preconditioning agents include 3-iodothyronamine (TlAM), 3- methylthyronamine, N-methyl-O-(p-trifluoromethyl)benzyl-tyramine hydrochloride, O- phenyl-3-iodotyramine hydrochloride or O-(p-Fluoro)phenyl-3-iodotyramine hydrochloride. In one specific example, the agent is TlAM.

[030] The thyronamine preconditioning agent is administered in one or more doses sufficient to elicit preconditioning, such as at least about 0.005 mg/kg. In certain embodiments, the agent is administered at a dose of at least about 0.02 mg/kg. Typically, no more than about 5 mg/kg of the agent is administered, such as about 0.2 mg/kg. Hence in certain embodiments the dosage range is 0.005-5 mg/kg, or 0.02-5 mg/kg, or 0.2-5 mg/kg.

[031] Another feature of this disclosure is the use of a thyronamine preconditioning agent in the preparation of a medicament for the induction of hypothermia to provide a prophylactic treatment of cellular injury, such as injury resulting from an excitotoxic, ischemic and/or hypoxic event. The preconditioning agent can be combined with other active agents (either in the composition or in methods of treatment). For example, the preconditioning agent can be administered in advance of a potential ischemic event, and another protective agent (such as osteopontin) can be administered at the time the actual injury occurs. In other examples, the preconditioning agent can be combined with a second agent designed to help treat the condition (such as vascular occlusion or hypertension) leading to the cellular injury. For example the second agent could be an anti-hypertensive (such as enalapril), a treatment for hypercholesterolemia (for example a lipid lowering drug such as Lipitor/atorvastin calcium), or an anticoagulant drug (such as a drug that inhibits platelet aggregation for example ticlopodine or aspirin).

Terms

[032] Unless otherwise explained, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinaiy skill in the art to which this disclosure belongs. Definitions of common terms in molecular biology may be found in Benjamin Lewin, Genes V, published by Oxford University Press, 1994 (ISBN 0-19-854287- 9); Kendrew et al. , (eds.), The Encyclopedia of Molecular Biology, published by Blackwell Science Ltd., 1994 (ISBN 0-632-02182-9); and Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by VCH Publishers, Inc., 1995 (ISBN 1-56081-569-8). [033] The singular terms "a," "an," and "the" include plural referents unless context clearly indicates otherwise. Similarly, the word "or" is intended to include "and" unless the context clearly indicates otherwise. The term "plurality" refers to two or more. It is further to be understood that all base sizes or amino acid sizes, and all molecular weight or molecular mass values, given for nucleic acids or polypeptides are approximate, and are provided for description. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of this disclosure, suitable methods and materials are described herein. The term "comprises" means "includes." The abbreviation, "e.g." is derived from the Latin exempli gratia, and is used herein to indicate a non-limiting example. Thus, the abbreviation "e.g." is synonymous with the term "for example."

[034] In order to facilitate review of the various embodiments of this disclosure, the following explanations of specific terms are provided:

[035] The phrase "excitotoxic injury" or "excitotoxic brain injury" refers to injury (including death), of neural cells, particularly neural cells of the brain, due to excessive stimulation of cell-surface receptors. Most commonly, excitotoxic injury is mediated through glutamate receptors, for example, by overactivation of N-methyl-d-aspartate (NMDA)-type glutamate receptors, resulting in excessive Ca²⁺ influx through the receptor's associated ion channel. Excitotoxic injury is believed to play a role in diverse conditions, including epilepsy, traumatic injury, and Alzheimer's disease. An excitotoxic event is an event that results in excessive and deleterious stimulation of cell surface receptors.

[036] The term "hypoxia" refers to a lack of oxygen. In a physiological context, the term hypoxia refers to an insufficiency of oxygen at a cellular, tissue or organismal level. Hypoxia can be caused by, for example, the reduction in partial pressure of oxygen (in the blood or in a tissue), inadequate oxygen transport (for example, due to a failure of oxygenated blood to reach a target tissue or cell), or the inability of the tissues to use oxygen. Hypoxia can result from an increased oxygen demand in tissue that is unable to provide a commensurate increased oxygen supply, for example because of vascular disease. The term "infarct" refers to cell or tissue death due to a localized lack of oxygen (hypoxia). A hypoxic event is an event that results in insufficiency of oxygen at a cellular, tissue or organismal level.

[037] Frequently, hypoxia is the result of "ischemia," the reduction in oxygenated blood flow to a target tissue or organ. An "ischemic event" is an event or occurrence that results in decreased blood flow to a cell, collection or group of cells, tissue, or organ. Ischemic events include vasoconstriction, thrombosis and embolism, resulting in reduced blood flow to a tissue or organ.

[038] The term "stroke" refers to an interruption of the blood supply to any part of the brain. A stroke can be due to an ischemic event (for example, occlusion of a blood vessel due to a thrombus or an embolism) or hemorrhage (for example, of a cerebral blood vessel).

[039] A subject is "at risk for" an ischemic or hypoxic event if there is an increased probability that the subject will undergo an ischemic or hypoxic event relative to the general population. Accordingly, risk is a statistical concept based on empirical and/or actuarial data. Commonly, risk can be correlated with one or more indicators, such as symptoms, signs, characteristics, properties, occurrences, events or undertakings, of a subject. For example, with respect to risk of stroke, indicators include but are not limited to high blood pressure (hypertension), atrial fibrillation, transient ischemic events, prior stroke, diabetes, high cholesterol, angina pectoris, and heart disease. More generally, risk indicators for hypoxic events include surgery, especially cardiovascular surgeries, such as endarterectomy, pulmonary bypass surgery or coronary artery bypass surgery. Additional risk factors or indicators include non-medical activities, such as motorcycle riding, contact sports and combat. Other risk factors are discussed herein, and yet more can be recognized by those of ordinary skill.

[040] The term "protect" with respect to an excitotoxic, ischemic or hypoxic event refers to the ability of composition or treatment regimen to prevent, reduce in severity, or otherwise lessen the effects of an excitotoxic, ischemic or hypoxic event at a cellular, tissue or organismal level. Methods for measuring severity of effects of an excitotoxic, ischemic or hypoxic event include neurological, including behavioral, indicia (e.g., ascertainable via neurological examination of a subject) as well as by evaluation of cellular and metabolic parameters. Certain examples of techniques for evaluating tissue damage include Computed Axial Tomography (CT scan, CAT scan); Magnetic Resonance Imaging (MRI scan, MR scan); Carotid Ultrasound, including Transcranial Doppler (TCD); Cerebral Angiography: (Cerebral arteriogram, Digital subtraction angiography [DSA]); Computed Tomographic Angiography: (CT-angiography, CT-A, CTA); Magnetic Resonance Angiography (MRA) and/or other diagnostic procedures known to those of ordinary skill in the art. One indication of neurological damage is the presence of an infarct detectable on a CT or MRI scan of the brain. [041] "Cytoprotection" refers to cellular protection, such as the protection provided by hypothermia against cellular injury induced by hypoxia (such as that caused by ischemia) or excitotoxic injury (such as that seen in stroke and many neurological diseases).

[042] A "subject" is a living multi-cellular vertebrate organism, a category that includes both human and veterinary subjects, including human and non-human mammals. In a clinical setting with respect to preconditioning against excitotoxic injury and/or hypoxia, a subject is usually a human subject, although veterinary subjects are also contemplated.

[043] A "neural cell" is any cell in a lineage that originates with a neural stem cell and includes a mature neuron. Thus, the term neural cell includes neurons (nerve cells) as well as their progenitors regardless of their stage of differentiation. In the context of an adult brain, neural cells are predominantly differentiated neurons. In contrast, a "non-neural cell" is a cell of a lineage other than a neural cell lineage, that is a lineage that does not culminate in the differentiation of a mature neuron. The non-neural cell may reside in the central nervous system (CNS), for example, in the brain (such as glial cells and immune system cells, such as B cells, dendritic cells, macrophages and microglia), or may exist in an organ outside the CNS, such as cardiac, skeletal or smooth muscle (a muscle cell), liver (a hepatic cell) or kidney (a renal cell) and so forth. Non-neural cells include cells of the immune system, regardless of whether they reside in the CNS or elsewhere in the body of the organism.

[044] "Neurological injury" refers to damage to neural tissue (such as the brain, spinal cord, or peripheral nerves) that impairs the function of the neural tissue. Examples of neurological injury include brain injury that leads to cognitive impairment or motor or sensory deficits.

[045] A "cytoprotective cytokine" is a soluble protein (or glycoprotein) involved in the regulation of cellular proliferation and function that acts to preserve cellular function and prevent (or reduce) death of a cell in response to a stressful or otherwise aversive stimulus. Cytoprotective cytokines include transforming growth factor β (TGF-β), tumor necrosis factor α (TNFα), and type I interferons, such as interferon β (IFNβ). A "neuroprotective cytokine" is a cytoprotective cytokine that acts to preserve cellular function and reduce cell death in neural cells.

[046] The term "medicament" is used interchangeably with the term "pharmaceutical composition." Such compositions are formulated for administration to human and/or animal (veterinary) subjects, and typically include one or more active component (such as one or more of the thyronamine preconditioning agents disclosed herein) as well as one or more additional components to facilitate administration to a subject, for the therapeutic or prophylactic treatment (prevention or reduction) of a condition or disease. The additional components can include pharmaceutically acceptable carriers, buffers or excipients. Pharmaceutically acceptable carriers, buffers and so forth, are well known in the art, and are described, e.g., in Remingtons Pharmaceutical Sciences, 19^th Ed., Mack Publishing Company, Easton, Pennsylvania, 1995.

[047] "Prophylactic" treatment refers to the treatment of a subject prior to the full manifestation of an event, condition or disease for the purpose of preventing or reducing the symptoms, signs or consequences of the event, condition or disease. Thus, in the context of the present disclosure, prophylactic treatment of ischemia or hypoxia refers to the treatment of a subject prior to the occurrence of an ischemic or hypoxic event (that is, prior to a first ischemic or hypoxic event, or prior to a subsequent ischemic or hypoxic event, or prior to the completion or culmination of an ongoing or recurrent ischemic or hypoxic event) and prior to the completion of the natural consequences and/or sequelae of the event. In some embodiments of the method disclosed herein, the prophylactic treatment occurs prior to the onset of hypoxia that can lead to neurological damage.

[048] The term "thyronamine preconditioning agent" refers to a thyroxine derivative compound that produces a cytoprotective (for example, neuroprotective) effect when administered to a subject prior to the onset of an excitotoxic, ischemic and/or hypoxic event. Thyronamine preconditioning agents are potent agonists of the TAARl receptor, and are characterized by the ability to induce hypothermia in vivo. Examples of thyronamine preconditioning agents include 3-iodothyronamine, 3-methylthyronamine, N-methyl-(9-(p- trifluoromethyl)benzyl-tyramine hydrochloride, O-phenyl-3-iodotyramine hydrochloride or O- (p-Fluoro)phenyl-3-iodotyramine hydrochloride. Structures of exemplary thyronamine preconditioning agents are illustrated in FIGS. IA-D and in Tables 1 and 2.

[049] With respect to thyronamine, "analog" or "functional analog" refers to a modified form of the respective thyronamine derivative in which one or more functional side or linking groups has been modified such that the analog retains substantially the same biological activity or improved biological activity as the unmodified thyronamine derivative in vivo and/or in vitro. [050] "Agonist" or "thyronamine agonist" refers to an endogenous or exogenous compound, substance or entity that has affinity for and stimulates physiologic activity at cell receptors normally stimulated by naturally-occurring substances, thus triggering a biochemical response characteristic of those receptors. As used herein, the term refers to a thyronamine derivative or analog, a suitable homolog, or a portion thereof, capable of promoting at least one of the biological responses normally associated with thyronamine. For example, treatment with a thyronamine agonist can result in lowered body temperature of a mammalian subject.

[051] "Receptor" refers to a molecule, a polymeric structure, or polypeptide in or on a cell that specifically recognizes and binds a compound acting as a molecular messenger, for example, neurotransmitter, hormone, lymphokine, lectin, or drug. A ligand is said to "activate" a receptor if the ligand binds to the receptor, and such binding results in the initiation of one or more signaling events, such as translocation or phosphorylation of the receptor and/or other signaling molecules.

[052] A "TAARl" receptor is trace-amine-associated receptor 1, for which the thyronamine preconditioning agents are ligands. Exemplary TAARl receptors are catalogued in GenBank, for example, the sequence of the mouse TAARl receptor can be found under GenBank reference numbers NP444435, AAI25371, AAI25369; the sequence of the rat TAARl receptor can be found under accession number NP599155; and the human TAARl receptor can be found under accession number AAIO 1826 (all as of the filing date of this disclosure).

[053] The term "acyl" refers group of the formula RC(O)- wherein R is an organic group.

[054] "Lower alkyl" refers to an optionally substituted, saturated straight or hydrocarbon having from about 1 to about 12 carbon atoms (and all combinations and subcombinations of ranges and specific numbers of carbon atoms therein), with from about 1 to about 8 carbon atoms, being preferred. Alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, t-butyl, n-pentyl, cyclopenlyl, isopentyl, neopentyl, n-hexyl, isohexyl, 3-methylpentyl, 2,2-dimethylbutyl, and 2,3-dimethylbutyl. Specifically included within the definition of "lower alkyl" are those aliphatic hydrocarbon chains that are optionally substituted.

[055] The term "cycloalkyl" refers to a non-aromatic carbon-based ring composed of at least three carbon atoms. Examples of cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and the like. [056] "Optional" or "optionally" means that the subsequently described event or circumstance may or may not occur, and that the description includes instances in which it does not.

[057] "Effective amount" refers to an amount of a compound that can be therapeutically (including prophylactically) effective to inhibit, prevent or treat the symptoms of particular disease, disorder or side effect.

[058] "Pharmaceutically acceptable" refers to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem complications commensurate with a reasonable benefit/risk ratio. Pharmaceutically acceptable carriers, excipients and diluents are known to those of ordinary skill in the art and are described, for example, in Remington 's Pharmaceutical Sciences, by E. W. Martin, Mack Publishing Co., Easton, PA, 19th Edition (1995), which describes compositions and formulations suitable for pharmaceutical delivery of the compounds herein disclosed.

[059] "In combination with," "combination therapy" and "combination products" refer, in certain embodiments, to the concurrent administration to a patient of the thyronamine preconditioning agents and at least one additional pharmaceutical (for example, therapeutic) agent. When administered in combination, each component can be administered at the same time or sequentially in any order at different points in time. Thus, each component can be administered separately but sufficiently closely in time so as to provide the desired therapeutic effect. In some embodiments, the thyronamine preconditioning agent is administered in combination with a therapeutic agent that reduces injury when administered during or following an excitotoxic, ischemic and/or hypoxic event.

[060] "Dosage unit" refers to physically discrete units suited as unitary dosages for the particular individual to be treated. Each unit can contain a predetermined quantity of active compound(s) calculated to produce the desired therapeutic effect(s) in association with the required pharmaceutical carrier. The specification for the dosage unit forms can be dictated by (a) the unique characteristics of the active compound(s) and the particular therapeutic effect(s) to be achieved, and (b) the limitations inherent in the art of compounding such active compound(s). [061] A "preconditioning dose" is a dose of an effective compound, or composition containing such a compound, that protects a cell against injury or death, for example injury or death due to an ischemic or hypoxic event. In certain examples disclosed herein, the preconditioning dose is an amount that is effective to induce hypothermia sufficient to provide a cytoprotective effect. The dosage of the effective compound or composition varies from compound to compound and between species. A suitable preconditioning dose for any compound can be determined empirically.

Preconditioning

[062] Exposure of cells to subthreshold levels (that is, at a level below that which causes injury) of a stressful (e.g., cytotoxic) stimulus can induce tolerance to subsequent events that would otherwise result in injury. This effect has been termed preconditioning, and is relevant to preventing or reducing injury due to cytotoxic insult such as hypoxia (e.g., due to ischemic events) in a variety of cell and tissue types, including neural cells, muscle cell (e.g., skeletal as well as cardiac muscle cells), kidney cells and liver cells.

[063] Preconditioning sometimes involves a fundamental change in the genomic program or response (that is, the pattern of gene expression produced in response) to excitotoxic, ischemic and/or hypoxic injury that shifts the outcome from cell death to cell survival (Stenzel-Poore et al, The Lancet, 362:1028-1037, 2003). This change in gene expression, or genomic reprogramming, in response to cytotoxic insults, such as excitotoxic, ischemic and/or hypoxic events, involves a pronounced suppression of gene expression (for example, of inflammatory cytokines, and certain ion channels and channel regulators, e.g., K⁺ and Ca⁺⁺ channels, such as glutamate receptors), which is ordinarily injurious. Such suppression contrasts sharply with the upregulation of mRNA by ischemic and/or hypoxic events without preconditioning. This change is not simply the lack of a response, but rather a reprogramming of the genomic response that involves the downregulation of genes that control metabolism, cell-cycle regulation, and, in neural cells, ion-channel activity. Additionally, in certain cells of the immune system, preconditioning elicits a shift from pro-inflammatory to anti-inflammatory cytokines.

[064] Preconditioning in the brain (that is, of neural cells) and other organs can be produced following hypothermia, that is, to a reduction in core body temperature. This effect is dependent on de novo protein synthesis, and involves changes in genomic programming associated with inflammation. Thyroxine preconditioning agents, such as the thyronamine derivatives 3-iodothyronamine (TlAM), 3-methylthyronamine, N-methyl-C>-(p- trifluoromethyl)benzyl-tyramine hydrochloride, O-phenyl-3-iodotyramine hydrochloride or O- (p-Fluoro)phenyl-3-iodotyramine hydrochloride, can favorably be used as preconditioning agents.

[065] The mechanism by which hypothermic preconditioning protects the brain is different than the mechanism by which acute administration of hypothermia protects the brain. Acute induction of hypothermia can directly protect neurons post-ischemia by reducing reaction rate by the QlO effect. The QlO effect refers to the factor by which biochemical reaction rate is increased for each 10°C increase in temperature. For each 1⁰C decrease in temperature there will be a 10% reduction in tissue metabolic requirements and free radical production. The QlO effect can benefit neurons when hypothermia is applied during or post ischemia but not when hypothermia is applied before ischemia because it is remote to the injury process. Instead the protective effect of hypothermic preconditioning appears to be reliant on cellular reprogramming and direct biochemical changes in the intracellular and extracellular milieu. Administration of thyronamine preconditioning agents as disclosed herein elicits effective multipathway reprogramming associated with hypothermic preconditioning.

[066] Following administration of a suitable thyronamine derivative (such as the thyronamine preconditioning agents disclosed herein), cytoprotection against cellular injury (such as that caused by excitotoxic, ischemic and hypoxic injury) typically begins within about 10-12 hours and lasts for up to several weeks, or more. In addition, protection can be extended by repeated administration of the agent.

Selecting Subjects at Risk for Cytotoxic Insult

[067] The methods disclosed herein are applicable to any cell types susceptible to ischemic and/or hypoxic injury, which are amenable to preconditioning. For example, neural cells (including, e.g., hippocampal neurons and cortical neurons), muscle cells (including cardiac and striated muscle cells), hepatic cells and renal cells can be protected against injury and death by administering a thyronamine preconditioning agent prior to the occurrence of an event capable of inducing the cellular injury, such as an excitotoxic, ischemic or hypoxic event. Thus, a thyronamine preconditioning agent (such as, 3-iodothyronamine, 3- methylthyronamine, N-methyl-(9-(p-trifluoromethyl)benzyl-tyramine hydrochloride, O-phenyl- 3-iodotyramine hydrochloride or 0-(p-Fluoro)phenyl-3~iodotyramine hydrochloride) can be administered to a subject that has been identified as having (e.g., diagnosed with) one or more risk factors indicative of an increased likelihood, relative to the general population or to a subject without the risk factor, of having an excitotoxic, ischemic and/or hypoxic event.

[068] Preconditioning with thyronamine derivatives is useful for inhibiting (including preventing) cellular injury due to excitotoxic, ischemic and/or hypoxic events associated with a wide variety of conditions with disparate etiologies and symptoms, including stroke, traumatic brain injury and Alzheimer's disease. For example, in addition to medical indications such as stroke or Alzheimer's disease, non-medical indicators of risk, pertaining to behaviors or activities that are statistically associated with an increased likelihood of injuries that can include a hypoxic component. Additionally, such conditions can also include an excitotoxic component. For example, traumatic brain injury (regardless of its cause) frequently involves an excitotoxic and/or a hypoxic component. Thus, participation in activities that increase the risk of traumatic brain injury is an indicator that can be used to select a subject for administration of a preconditioning agent (such as a thyronamine derivative). Such activities include, for example, motorcycle riding, motor vehicle racing, skiing, contact sports (such as, football, hockey, rugby, soccer, lacrosse, martial arts, boxing and wrestling), and the like. Additionally, impacts or wounds resulting from gunshot or explosives frequently cause traumatic brain injury. Accordingly, activities that are associated with an increased risk of gunshot wounds or injury caused by explosive devices (for example, in combat situations) are an indicator of risk that can be used to select a subject for treatment with a thyronamine preconditioning agent according to the methods disclosed herein.

[069] Hypoxia is typically associated with ischemic events in the CNS or elsewhere in the cardiovasculature, (such as cerebrovascular ischemia, or stroke, myocardial ischemia due to narrowing or blockage of the vessels of the heart, iatrogenic ischemia, due to surgical procedures, and the like). For example, more than a quarter of patients that undergo coronary artery bypass graft (CABG) surgery suffer cognitive deficits following surgery. Such individuals are at high risk for neurological injury, such as that caused by an excitotoxic, ischemic and/or hypoxic event, and thus are subjects who can benefit from prophylactic administration of a thyronamine preconditioning agent prior (for example, between 24 hours and 1 week prior) to undergoing CABG surgery.

[070] In addition, hypoxia can occur in utero due to conditions such as inadequate placental function (for example, due to abruptio placentae), preeclamptic toxicity, prolapse of the umbilical cord, or complications from anesthetic administration. Additionally, injury by some hypoxic events (such as strokes) involves an excitotoxic component as well as a hypoxic component.

[071] Similarly, various cardiovascular signs and symptoms, such as atrial fibrillation, angina pectoris, hypertension, transient ischemic episodes and prior stroke, are all indicators of risk (or risk factors) that can be used to select a subject for administration of a thyroxine derivative, such as the thyronamine preconditioning agents (such as, 3-iodothyronamine, 3- methylthyronamine, N-methyl-C>-(p-trifluoromethyl)benzyl-tyramine hydrochloride, O-phenyl- 3-iodotyramine hydrochloride or 0-(p-Fluoro)phenyl-3-iodotyramine Hydrochloride, for example, TlAM) disclosed herein. Similarly, surgical procedures, especially those specifically involving the cardiovascular system, such as endarterectomy, pulmonary bypass and coronary artery bypass surgeries, are indicators of risk that can be used to select a subject for administration of a preconditioning agent. The presence of atheromatous plaques in a carotid artery (which is often indicated by partial occlusion of a carotid artery on a Doppler ultrasound scan) are also risk factors associated with a potential ischemic event that can be caused by rupture and/or dislodgement of a portion of the plaque that can cause thromboembolic occlusion of the neurovasculature.

Thyroxine derivatives

[072] Thyronamines are analogs of the thyroid hormone thyroxine (T₄). Disclosed herein are thyronamines and thyronamine analogs for use as preconditioning agents. In certain embodiments such preconditioning agents exert a hypothermic effect, such as by activating TAAR. Certain preconditioning agents have the formula

[073] wherein X represents -CR₂-, -C(O)CH₂-, -(CH₂)_H1-;

[074] each R independently is selected from H and lower alkyl;

[075] m is from 0 to 4;

[077] Y is -NR¹R², or OR³;

[078] R¹ and R² independently are H, lower alkyl, ben:zyl, or together form a cycloalkyl group;

[079] R³ is H, lower alkyl or acyl;

[080] R⁴ and R⁵ independently are H, cyano, halo, lower alkyl, fluoroalkyl, or -OR¹¹;

[081] R¹¹ is H, acyl or lower alkyl;

[082] R⁶, R⁷, R⁸, R⁹ and R¹⁰ independently are selected from H, halogen, lower alkyl, haloalkyl nitro, or -OR¹²; and

[083] R¹² is H, acyl or lower alkyl.

[084] In other embodiments, preconditioning agents are provided of the formula:

wherein variable groups X, R , R and R >4- rR> 10 are selected from those groups set forth above. In particular embodiments, with continued reference to the formula above, at least one of R⁴, R⁵, R⁶ and R¹⁰ is an iodo group, for example, R⁴ and/or R⁵ may be iodo. In additional compounds at least one of R⁴-R¹⁰ is acyloxy (-OC(O)R), alkoxy, or hydroxy. For example, embodiments of preconditioning agents are represented by the formulas

or

wherein variable groups X, R¹, R² and R⁴, R⁵ and R^!2 are selected from the groups set forth above.

[085] The results of in vitro assessment of exemplary preconditioning compounds having the formulas above are recorded below in Table 1 :

[086] Table 1: Activation of TAARl by Exemplary Preconditioning Agents

[087] In other embodiments the preconditioning agents used have the formula

[088] wherein the variable groups X, Y and R⁴-R¹⁰ are as set forth above. Particular examples of preconditioning agents according to this formula were evaluated for hypothermic potential in vitro and the results are recorded in Table 2:

[090] Methods for synthesizing thyronamines, including thyronamine preconditioning agents, are described in detail in U.S. Patent No. 6,979,750, and in Hart et al, J. Med. Chem., 49: 1101-1112, 2006, the disclosures of which are incorporated herein by reference to the extent that they are not inconsistent with the present disclosure. In brief, thyronamine derivatives can be prepared from tyramine using copper mediated coupling of a boronic acid or analog and the appropriate protected phenol. Variations in the side chains (e.g., at Re) can be made by utilizing the appropriately protected boronic acid, or by other methods known to those of skill in the art. U.S. Patent No. 6,979,750 provides several alternative methods for preparing thyronamine derivatives, including thyronamine preconditioning agents with one or more R group substitutions. Specific examples of R groups include amine groups, and salts thereof, halyl (e.g., I, F), lower alkyl groups, e.g., methyl groups, as well as substituted alkyls, including haloalkyl groups.

[091] These methods are suitable for producing thyronamine preconditioning agents on a large scale, and at a purity suitable for therapeutic use. Thus, the thyronamine preconditioning agents can be synthesized and purified to a substantially pure form that is substantially devoid of organic impurities, such as unreacted starting reagents, unreacted intermediate compounds, and other compounds other than the desired thyronamine derivative(s). For example, the thyronamine preconditioning agent can be at least 75% pure, such as at least about 80% pure, or at least about 85% pure. In certain instances, the thyronamine derivative is at least about 90% pure, or even about 95% or 98% pure. For some applications disclosed herein, the thyronamine derivative can be greater than 99% pure, such as 100% pure.

[092] The term thyronamine preconditioning agent includes in addition to the bioactive thyroxine derivatives expressly described, stereoisomers, prodrugs, pharmaceutically acceptable salts, hydrates, solvates, acid salt hydrates, N-oxide or isomorphic crystalline forms, etc. These thyronamine derivatives include compounds in which the parent compound is modified by making acid or base salts thereof. Examples of stereoisomer, prodrug, pharmaceutically acceptable salt, hydrate, solvate, acid salt hydrate, N-oxide or isomorphic crystalline form thereof include, but are not limited to, mineral or organic acid salts of basic residues such as amines; alkali or organic salts of acidic residues such as carboxylic acids; and the like. The stereoisomer, prodrug, pharmaceutically acceptable salt, hydrate, solvate, acid salt hydrate, N-oxide or isomorphic crystalline form thereof include the conventional non-toxic salts or the quaternary ammonium salts of the parent compound formed, for example, from non-toxic inorganic or organic acids. For example, such conventional non-toxic salts include those derived from inorganic acids such as hydrochloric, hydrobromic, sulfuric, sulfamic, phosphoric, nitric and the like; and the salts prepared from organic acids such as acetic, propionic, succinic, glycolic, stearic, lactic, malic, tartaric, citric, ascorbic, pamoic, maleic, hydroxymaleic, phenylacetic, glutamic, benzoic, salicylic, sulfanilic, 2-acetoxybenzoic, fumaric, toluenesulfonic, methanesulfonic, ethane disulfonic, oxalic, isethionic, and the like.

[093] These physiologically acceptable salts are prepared by methods known in the art, e.g., by dissolving the free amine bases with an excess of the acid in aqueous alcohol, or neutralizing a free carboxylic acid with an alkali metal base such as a hydroxide, or with an amine.

[094] Compounds described herein throughout, can be used or prepared in alternate forms. For example, many amino-containing compounds can be used or prepared as an acid addition salt. Often such salts improve isolation and handling properties of the compound. For example, depending on the reagents, reaction conditions and the like, compounds as described herein can be used or prepared, for example, as their hydrochloride or tosylate salts. Isomorphic crystalline forms, all chiral and racemic forms, N-oxide, hydrates, solvates, and acid salt hydrates, are also contemplated to be within the scope of the present compositions and methods.

[095] Certain acidic or basic compounds can exist as zwitterions. All forms of the compounds, including free acid, free base and zwitterions, are contemplated to be within the scope of the present compositions and methods. It is well known in the art that compounds containing both amino and carboxyl groups often exist in equilibrium with their zwitterionic forms. Thus, any of the compounds described herein throughout that contain, for example, both amino and carboxyl groups, also include reference to their corresponding zwitterions.

[096] Also included are thyronamine derivatives that are prodrugs. A prodrug is a compound specifically designed to maximize the amount of the thyronamine active species that reaches the desired site of reaction, which is inactive or minimally active for the activity desired, but through biotransformation is converted into the biologically active thyronamine derivative.

[097] One of skill in the art will readily appreciate that when any variable occurs more than one time in any constituent or in any formula, its definition in each occurrence is independent of its definition at every other occurrence. Combinations of substituents and/or variables are permissible only if such combinations result in stable compounds.

[098] In certain embodiments, thyronamine preconditioning agents are selected to induce profound hypothermia in a subject. For example, TlAM is shown herein to induce a lowering of a subject's temperature (e.g., as measured using a rectal or infrared thermometer) by at least 6⁰C, or more. For example, as demonstrated herein, TlAM induces a reduction of temperature (as measured in a mouse model) from 37°C to 31°C, which is sustained for a prolonged period of up to six or more hours. Thus, a preconditioning agent can be selected that reduces a subject's body temperature by at least 6⁰C, to at least 31°C, and/or to an equal or greater extent (that is, to a lower temperature) than TlAM.

[099] In a particular example, TlAM is the thyronamine preconditioning agent. Additional example of thyronamine derivatives suitable as preconditioning agents that reduce a subject's temperature by at least 6⁰C (that is, to an extent equal to or greater than TlAM) include 3- methylthyronamine, N-methyl-O-(p-trifluoromethyl)benzyl-tyramine hydrochloride, O-phenyl- 3-iodotyramine hydrochloride and <9-(p-Fluoro)phenyl-3-iodotyramine Hydrochloride. Additional thyronamine derivatives that are suitable as thyronamine preconditioning agents can easily be identified (and novel agents can be synthesized) that induce hypothermia to an equivalent level and for an equivalent duration as TlAM.

Pharmaceutical compositions

[0100] Thyronamine preconditioning agents can be administered to a subject, such as a human subject, as disclosed herein or in the form of a stereoisomer, prodrug, pharmaceutically acceptable salt, hydrate, solvate, acid salt hydrate, N-oxide or isomorphic crystalline form thereof, or in the form of a pharmaceutical composition where the compound is mixed with suitable carriers or excipient(s) in a therapeutically effective amount, for example, a preconditioning dose to protect against excitotoxic, ischemic and/or hypoxic injury.

[0101] The thyronamine derivatives and analogs and pharmaceutical compositions described herein can be administered by a variety of routes. Suitable routes of administration can, for example, include oral, nasal, transmucosal (e.g., rectal), or intestinal administration; parenteral delivery, including intramuscular, subcutaneous, intramedullary injections, as well as intravenous, intrathecal, intraventricular, spinal, epidural, or intraperitoneal injections. Alternatively, one can administer the compound in a local rather than systemic manner, for example via injection of the compound directly into the subject at a specific site, often in a depot or sustained release formulation. Furthermore, one can administer the compound in a targeted drug delivery system, for example, in a liposome coated vesicle. The liposomes can be targeted to and taken up selectively by the tissue of choice.

[0102] In one embodiment, the thyronamine preconditioning agents described herein are administered orally or nasally.

[0103] The pharmaceutical compositions (medicaments) described herein can be manufactured in a manner that is itself known, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes. Pharmaceutical compositions for use as described herein can be formulated in conventional manner using one or more physiologically acceptable carriers comprising excipients and auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceutically.

[0104] Proper formulation is dependent upon the route of administration chosen. For injection, the agents can be formulated in aqueous solutions, e.g., in physiologically compatible buffers such as Hanks' solution, Ringer's solution, or physiological saline buffer. For transmucosal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art. For example, for oral administration, the compounds can be formulated readily by combining with pharmaceutically acceptable carriers that are well known in the art. Such carriers enable the compounds to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions and the like, for oral ingestion by a patient to be treated. Pharmaceutical preparations for oral use can be obtained by mixing the compounds with a solid excipient, optionally grinding a resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries, if desired, to obtain tablets or dragee cores.

[0105] Suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl- cellulose, sodium carboxymethylcellulose, and/or polyvinylpyrrolidone (PVP). If desired, disintegrating agents can be added, such as the cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate. Dragee cores are provided with suitable coatings. For this purpose, concentrated sugar solutions can be used, which can optionally contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures. Dyestuffs or pigments can be added to the tablets or dragee coatings for identification or to characterize different combinations of active compound doses.

[0106] Pharmaceutical preparations which can be used orally include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol. The push-fit capsules can contain the active ingredients in admixture with filler such as lactose, binders such as starches, and/or lubricants such as talc or magnesium stearate and, optionally, stabilizers. In soft capsules, the active compounds can be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols. In addition, stabilizers can be added. All formulations for oral administration should be in dosages suitable for such administration. For buccal administration, the compositions can take the form of tablets or lozenges formulated in conventional manner. For administration by inhalation, the compounds for use are conveniently delivered in the form of an aerosol spray presentation from pressurized packs or a nebuliser, with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In the case of a pressurized aerosol the dosage unit can be determined by providing a valve to deliver a metered amount. Capsules and cartridges of e.g. gelatin for use in an inhaler or insufflator can be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.

[0107] The compounds can be formulated for parenteral administration by injection, e.g., by bolus injection or continuous infusion. Formulations for injection can be presented in unit dosage form, e.g., in ampules or in multi-dose containers, with an added preservative. The compositions can take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and can contain formulatory agents such as suspending, stabilizing and/or dispersing agents. Pharmaceutical formulations for parenteral administration include aqueous solutions of the active compounds in water-soluble form. Additionally, suspensions of the active compounds can be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes. Aqueous injection suspensions can contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Optionally, the suspension can also contain suitable stabilizers or agents which increase the solubility of the compounds to allow for the preparation of highly concentrated solutions. Alternatively, the active ingredient can be in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.

[0108] The compounds can also be formulated in rectal compositions such as suppositories or retention enemas, e.g., containing conventional suppository bases such as cocoa butter or other glycerides. In addition to the formulations described previously, the compounds can also be formulated as a depot preparation. Such long acting formulations can be administered by implantation (for example subcutaneously or intramuscularly) or by intramuscular injection. Thus, for example, the compounds can be formulated with suitable polymeric or hydrophobic materials (for example as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt.

[0109] A suitable pharmaceutical carrier for hydrophobic compounds is a cosolvent system comprising benzyl alcohol, a nonpolar surfactant, a water-miscible organic polymer, and an aqueous phase. The cosolvent system can be the VPD co-solvent system. VPD is a solution of 3% (w/v) benzyl alcohol, 8% (w/v) of the nonpolar surfactant polysorbate 80, and 65% (w/v) polyethylene glycol 300, made up to volume in absolute ethanol. The VPD co-solvent system (VPD: 5W) consists of VPD diluted 1: 1 with a 5% (w/v) dextrose in water solution. This cosolvent system dissolves hydrophobic compounds well, and itself produces low toxicity upon systemic administration. Naturally, the proportions of a co-solvent system can be varied considerably without destroying its solubility and toxicity characteristics. Furthermore, the identity of the co-solvent components can be varied: for example, other low-toxicity nonpolar surfactants can be used instead of polysorbate 80; the fraction size of polyethylene glycol can be varied; other biocompatible polymers can replace polyethylene glycol, e.g. polyvinyl pyrrolidone; and other sugars or polysaccharides can substitute for dextrose.

[0110] Alternatively, other delivery systems for hydrophobic pharmaceutical compounds can be employed. Liposomes and emulsions are well known examples of delivery vehicles or carriers for hydrophobic drugs. Certain organic solvents such as dimethylsulfoxide also can be employed, although usually at the cost of greater toxicity.

[0111] Additionally, the compounds can be delivered using a sustained-release system, such as semipermeable matrices of solid hydrophobic polymers containing the therapeutic agent. Various types of sustained-release materials have been established and are well known by those skilled in the art. Sustained-release capsules can, depending on their chemical nature, release the compounds for a few weeks up to over 100 days. The pharmaceutical compositions also can comprise suitable solid or gel phase carriers or excipients. Examples of such carriers or excipients include but are not limited to calcium carbonate, calcium phosphate, various sugars, starches, cellulose derivatives, gelatin, and polymers such as polyethylene glycols.

[0112] Pharmaceutically acceptable carriers are determined in part by the particular composition being administered, as well as by the particular method used to administer the composition. Accordingly, there is a wide variety of suitable formulations of pharmaceutical compositions for administering the iodo-thyronamine (see, e.g., Remington 's Pharmaceutical Sciences, by E. W. Martin, Mack Publishing Co., Easton, PA, 19th Edition (1995), incorporated herein by reference). The pharmaceutical compositions generally comprise a differentially expressed protein, agonist or antagonist in a foπn suitable for administration to a subject. The pharmaceutical compositions are generally foπnulated as sterile, substantially isotonic and in full compliance with all Good Manufacturing Practice (GMP) regulations of the U.S. Food and Drug Administration.

[0113] Pharmaceutical compositions suitable for use include compositions wherein the thyronamine preconditioning agents are contained in a therapeutically effective amount. Determination of an effective amount is well within the capability of those skilled in the art, especially in light of the detailed disclosure provided herein. For any compound used in the present method, a therapeutically effective dose can be estimated initially from cell culture assays. For example, a dose can be formulated in animal models to achieve a circulating concentration range that includes the LD₅₀ as determined in cell culture (i.e., the concentration of test compound that is lethal to 50% of a cell culture) or the LDioo as determined in cell culture (i.e., the concentration of compound that is lethal to 100% of a cell culture). Such information can be used to more accurately determine useful doses in humans.

[0114] Using these initial guidelines one having ordinary skill in the art could determine an effective dosage in humans. Initial dosages can also be estimated from in vivo data, such as in an animal model of stroke. One having ordinary skill in the art could readily optimize administration to humans based on this data. Dosage amount and interval can be adjusted individually to provide plasma levels of the active compound which are sufficient to maintain therapeutic effect. Therapeutically effective serum levels can be achieved by administering multiple doses each day. In cases of local administration or selective uptake, the effective local concentration of the drug can not be related to plasma concentration. One having skill in the art will be able to optimize therapeutically effective local dosages without undue experimentation. The amount of composition administered will, of course, be dependent on the subject being treated, on the subject's weight, the severity of the affliction, the manner of administration and the judgment of the prescribing physician. The therapy can be repeated intermittently, for example, in subjects at ongoing risk for an excitotoxic, ischemic and/or hypoxic event, or in subjects with a neurodegenerative disorder having an excitotoxic component, such as Alzheimer's disease.

[0115] Moreover, due to its apparent nontoxicity, the thyronamine preconditioning agent can be provided alone or in combination with other drugs, such as for example, antiinflammatories, antibiotics, corticosteroids, vitamins and the like. In some embodiments, the thyronamine preconditioning agent is administered prior to an excitotoxic, ischemic and/or hypoxic event as disclosed herein, and an additional pharmaceutical agent is administered during or following the event. The additional agent can also be a thyronamine derivative (such as the same thyronamine derivative as the thyronamine preconditioning agent, or an analog thereof), or the additional agent can be another compound that has a neuroprotective effect with respect to excitotoxic, ischemic and/or hypoxic injury (for example, endotoxin, CpG oligonucleotides, osteopontin). Synergism between the thyronamine derivatives described herein (or analogs) and other drugs can occur. In addition, possible synergism between a plurality of preconditioning agents can occur.

[0116] The typical daily dose of a pharmaceutical composition of thyronamine preconditioning agents varies according to individual needs, the condition to be treated and with the route of administration. Suitable doses are in the general range of from 0.001 to 10 mg/kg bodyweight of the recipient per day. The particular activity encountered at a particular dose will depend on the nature of the pharmaceutical composition of thyronamine derivatives and analogs used.

[0117] The pharmaceutical composition of thyronamine preconditioning agents can be in unit dosage form, for example, a tablet or a capsule so that the patient can self-administer a single dose, hi general, unit doses contain in the range of from 0.05—100 mg of a compound of the pharmaceutical composition of thyronamine derivatives and analogs. Unit doses contain from 0.05 to 10 mg of the pharmaceutical composition. The active ingredient can be administered from 1 to 6 times a day. Thus daily doses are in general in the range of from 0.05 to 600 mg per day. In an embodiment, daily doses are in the range of from 0.05 to 100 mg per day or from 0.05 to 5 mg per day. The dosage can vary within this range depending upon the dosage form employed and the route of administration utilized. The exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition. (See, e.g., Fingl et al., 1975, in: The Pharmacological Basis of Therapeutics, Ch. 1, p.l). Favorably, a thyronamine preconditioning agent is selected that has no or minimal toxicity at preconditioning dosage. One of the advantages, among others, of using the thyronamine derivatives and analogs described herein as preconditioning agents is their lack of toxicity at doses sufficient to elicit a preconditioning effect. For example, it has been found that repeated intraperitoneal doses of 75 mg/kg or TlAM produced no ill effects in mice. Since the i.v. serum half-life (tjβ) of TlAM is about 2-2.5 hours, repeated (e.g., daily, biweekly, or other interval of less than or up to about 1 week) dosages of the iodo-thyronamine derivatives described herein without ill effects is predictable.

EXAMPLES Example 1: In Vitro cAMP Assays.

[0118] HEK293 cells stably transfected with either rTAARl or mTAARl were harvested in Krebs-Ringer-HEPES buffer (KRH) and preincubated in KRH with 200 μM 3-isobutyl-l- methylxanthine (IBMX). Cells were incubated in KRH with 100 μM IBMX with the test compound, forskolin (10 μM), or vehicle (DMSO) for 1 h at 37 ⁰C (400 μL total volume). The cells were then boiled for 20 min after adding an equal volume of 0.5 mM sodium acetate buffer and centrifuged to remove cellular debris. An aliquot of the resulting extract (200 μL) was analyzed for cAMP content using competitive binding of [3HJcAMP to a cAMP binding protein (Diagnostic Products Corp., Los Angeles, CA). Data were reported relative to the forskolin control and expressed as %cAMP. Concentration-response curves were plotted, and EC50 values were calculated with Prism software (GraphPad, San Diego, CA). The R2 values in all cases were greater than 0.9. Experiments were run in triplicate and the standard error of the mean (SEM) was less than 0.1 log units from the average EC50 value in all cases.

Example 2: Acute administration of thyronamine derivates is neuroprotective

[0119] To demonstrate the efficacy of thyronamine derivatives as cryogens for the treatment of stroke, exemplary thyronamine derivatives, TlAM and TOAM, were administered in a mouse model of ischemia.

[0120] To first evaluate whether acute administration of thyronamine derivatives confers neuroprotection against ischemia and/or hypoxia associated with stroke injury, TlAM and TOAM were administered 1 hour following experimentally induced ischemia. [0121] TlAM and TOAM were obtained by chemical synthesis as described in U.S. Patent No. 6,979,750, and dissolved in 75% saline/25% DMSO.

[0122] C57BL/6 mice (male, 8-10 wks) were obtained from Jackson laboratories. All procedures met NIH guidelines with the approval of the Oregon Health & Science University Institutional Animal Care and Use Committee.

[0123] Temperature was measured using a non-invasive infrared measurement strategy. The repeated use of rectal probes is an uncomfortable and stressful procedure that can cause mucosal tearing leading to septicemia and death. As an alternative means of monitoring mouse temperature a Raytek MX2 infrared thermometer (emissivity set to 0.98) was employed using the procedure first published by Warn et al. (Warn et ah, Lab Anim., 37: 126-131, 2003). This thermometer proved reliable over a range of temperatures. When readings were compared from recently sacrificed mice (n=8) a high correlation was found between the infrared thermometer and a rectal thermometer, with an average difference in recorded temperature of 0.48⁰C.

[0124] For in vivo experiments a dose of 50mg/kg of TlAM and TOAM was selected based on the finding that this dose is sufficient to cause a level and duration of hypothermia that is protective in experiments using physical hypothermia. This dose of TlAM and TOAM is well tolerated, causing a temporary reduction in spontaneous activity in injected mice but no other discernable behavioral change. Table 2 shows behavioral scoring for 48 hours post injection of TlAM and TOAM. Mice were i.p. injected with vehicle, 50mg/kg TlAM or 50mg/kg TOAM and monitored hourly for the first 6 hours post injection (Table IA) and at 24 and 48 hours post injection (Table IB). (N=8 each group).

Table 2A. Behavioral scorin for hours 1-6 post injection.

Table 2B. Behavioral scoring for hours 24 and 48 post injection.

[0125] Cerebral focal ischemia was induced by middle cerebral artery occlusion (MCAO) as previously published (Clark et al., Neurol. Res., 19:641-648, 1997). Briefly, mice were anesthetized by halothane inhalation (4%/ 1 O2) and maintained with 1.5%/ 1 O2. The middle cerebral artery was blocked by threading silicone-coated 7-0 monofilament nylon surgical suture through the external carotid to the internal carotid, blocking its bifurcation into the MCA and anterior cerebral artery. The filament was maintained intraluminally for 60 min while the mice were maintained under anesthesia. The filament was then removed, thereby restoring blood flow. Cerebral blood flow (CBF) was monitored throughout the surgery by laser Doppler flowmetry (Periflow 5000; Perimed, Sweden). Body temperature was monitored throughout. Post surgery, mice had free access to soft food and were sacrificed after 24 hours.

[0126] Mice were i.p administered lOOμl of vehicle (75%saline/25%DMSO), TlAM (50mg/kg) or TOAM (50mg/kg) 1 hour after the end of ischemia and placed in a cage on an adjustable heating pad set to its lowest setting (Sunbeam Heating Pad). At this setting the temperature in the cage was 28⁰C, allowing the temperature of the drug treated animals to fall. For the TlAM and TOAM "hypothermia blocked" group of animals, mice were kept in a separate cage on an adjustable heating pad with the setting of the pad continually adjusted to keep the drug treated animals at the same temperature as the vehicle injected animals. Temperature was monitored by infrared thermometer.

[0127] Mice were deeply anesthetized with isoflurane, then perfused via the ascending aorta with heparinized saline (2 U/ml), at a flow rate of 9 ml/ min. Brains were rapidly removed, placed on a tissue slicer (Stoelting, Wood Dale, IL, USA) and covered with soft agarose. The olfactory bulb and cerebellum were removed and discarded. The remaining brain was sectioned into 1 mm slices beginning from the rostral end. To visualize the region of infarction, sections were placed in 1.5 % 2,3,5-triphenyltetrazolium chloride (TTC) in 0.9 % phosphate-buffered saline and stained for 15 min at 37°C ²⁶. After staining, the sections were transferred to 10 % paraformaldehyde. Images of the sections were scanned, and the area of the infarct and the ipsilateral hemisphere were measured by a technician blind to treatment group using NIH image 1.62. The measurements were multiplied by the section thickness (1 mm) and then summed over the entire brain to yield volume measurements. The percent infarct was calculated as: {Infarct Volume)l{Ipsilateral Hemisphere Volume) x 100. Data are shown as means ± SD of n determinations. Data from cell viability assays and in vivo stroke experiments were analyzed by one-way ANOVA followed by Bonferroni's multiple comparison test, using Graphpad Prism version 4.0 (Graphpad software, San Diego, CA, USA).

[0128] Injection of TlAM and TOAM caused an immediate reduction in temperature with temperature reaching as low as 31⁰C within 15 minutes of injection (FIG. 2A). Temperature reduction was in the absence of shivering or piloerection and by 24 hours post injection temperature had returned to the same level as vehicle injected mice. The hypothermic effect of TlAM and TOAM was equivalent, even though previous experiments on non-stroked mice have shown that TlAM is a more potent inducer of hypothermia than TOAM³. A possible explanation for this discrepancy is that following stroke mice become naturally hypothermic (as can be seen in the vehicle injected animals in Fig 2a and 4a) and this may alter the pharmacokinetics of TlAM and TOAM. Animals administered TlAM and TOAM had significantly reduced infarct volumes compared to control injected mice, showing a 35% and 32% reduction respectively (FIG. 2B). To determine if the protective effects of TlAM and TOAM were dependent on the hypothermic effects of the drugs, separate groups of animals were injected with TlAM or TOAM and maintained on an adjustable heating pad. The temperature of the mice was monitored hourly and the setting of the heating pad adjusted to maintain the mice at the same temperature as the vehicle injected animals. When the hypothermic effects of TlAM and TOAM were blocked the protective effects of these drugs was lost (FIG. 2B).

Example 3: Preconditioning with thyronamine derivatives

[0129] To demonstrate the efficacy of thyronamine derivatives as preconditioning agents, the exemplary derivatives TlAM and TOAM were administered 2 days prior to ischemia. Mice were i.p injected with lOOμl vehicle, TlAM (50mg/kg) or TOAM (50mg/kg) 2 days prior to MCAO. Following injection mice were maintained at ambient temperature (22⁰C) and temperature was recorded for 24 hours post injection as described in Example 1. For the TlAM "hypothermia blocked" group of animals, mice were kept in a separate cage on an adjustable heating pad with the setting of the pad continually adjusted to keep the temperature of the mice as close as possible to the vehicle treated mice. Temperature was monitored by infrared thermometer. Following surgery all preconditioned mice were kept on a heating pad set to low (the temperature in the cage was 28⁰C) and temperature was recorded every hour for at least 6 hours post surgery as well at 24 hours post surgery.

[0130] Following injection of TlAM and TOAM mice became hypothermic within 15 minutes (FIG. 3A). The recovery to normothermia of mice injected with TlAM was more prolonged than mice injected with TOAM. Preconditioning with TlAM reduced infarct volume by 34% compared to vehicle treated animals. Preconditioning with TOAM was less effective (FIG. 3B). Protection conferred by TlAM and TOAM appears to correlate with the duration of hypothermia induced with each drug. To determine if the protection observed with TlAM preconditioning could be attributed to hypothermia, we blocked the hypothermic effect of TlAM in a separate group of animals. In these animals the protective effect of the drug was lost, indicating that the protection seen with TlAM preconditioning is due to hypothermia.

Example 4: Neuroprotection by TOAM and TlAM is a systemic effect

[0131] To confirm that the acute neuroprotective effects of TlAM and TOAM are due to hypothermia, the ability of thyroxine derivatives to protect cells in vitro was evaluated. Following isoflurane anesthesia cerebral cortices were dissected from El 6 C57BL/6 mice and incubated in 0.05% trypsin-EDTA for 15 min at 37⁰C. Tissue was then triturated and cells plated on poly-L-ornithine coated 96-well plates or 25mm x 25mm glass coverslips at a density of 1 x 10⁵ per well or 10⁶ per coverslip. Cells were cultured in Neurobasal medium supplemented with L-glutamine and B27. Cultures consisted of 50-60% neurons as assessed by NeuN vs. GFAP staining on glass coverslips. Oxygen and glucose deprivation was performed on day 7 of culture.

[0132] Oxygen and glucose deprivation (OGD) was performed by washing cells with phosphate buffered saline (PBS) (0.5 mM CaCl₂, 1.0 mM MgCl₂; pH 7.4) and placing them in an anaerobic chamber for 180 min (Coy Laboratories, 85 % N₂, 5% H₂, 10 % CO₂; 35 ⁰C). OGD was terminated by removing cells from the chamber, replenishing with media and replacing them back into the normoxic incubator. A peptide including amino acids 109-153 of the osteopontin protein (GENBANK accession no. CAA36132), which confers neuroprotection was used as a positive control (Invitrogen, Carlsbad, CA, USA, dose: 5nM).

[0133] Cell viability assays were performed 24 hours post oxygen and glucose deprivation. 20μl of MTT (5mg/ ml dissolved in PBS) were added to each well of 96 well plates and the plates incubated for 1 hour at 37⁰C. The media was then removed from each well and the cells permeabilized with lOOμl DMSO. Cell viability was determined by reading plates at a wavelength of 550nm and comparing plates that had undergone OGD to plates that had only had media replaced (sham wash plates).

[0134] Primary mouse neuronal cultures were exposed to 3 hours of oxygen and glucose deprivation and different doses of TlAM and TOAM (5nM-500μM) were administered immediately post OGD. In the setting of cell culture TlAM and TOAM are unable to induce hypothermia, thus any observed neuroprotection would be the result of a direct effect. No protective dose of TlAM or TOAM was identified although higher doses of TlAM and TOAM were cytotoxic (FIG. 4A). This suggests that acute administration of TlAM and TOAM does not confer neuroprotection via a hypothermia independent mechanism. This experiment was repeated in primary rat neuronal cultures and the same result was obtained.

[0135] To further test the hypothesis that the preconditioning neuroprotective effect of TlAM is due to hypothermia we tested whether preconditioning administration of TlAM or TOAM could protect against ischemic injury in vitro. Primary mouse neuronal cultures were pretreated with doses of TlAM and TOAM ranging from 5nM to 500μM, 2 days prior to 3 hours of oxygen and glucose deprivation. No protective dose of TlAM or TOAM was identified (FIG. 4B). This experiment was repeated in primary rat neuronal cultures and the same result was obtained.

Example 5: Synthesis of Exemplary Preconditioning Agents

[0136] This example describes general procedures and exemplary specific syntheses for the disclosed preconditioning agents.

General Methods:

[0137] IH and 13C NMR spectra were taken on a Varian 400 (400 and 100 MHz, respectively). Data reported were calibrated to internal TMS (0.0 ppm) for all solvents unless otherwise noted and are reported as follows: chemical shift, multiplicity (app, apparent; par obsc, partially obscured; ovrlp, overlapping; br, broad; s, singlet; d, doublet; t, triplet; q, quartet; and m, multiplex), coupling constant, and integration. Inert atmosphere operations were conducted under argon passed through a Drierite drying tube in flame-dried or oven-dried glassware unless otherwise noted. Anhydrous THF, DCM, diethyl ether, pyridine, and diisopropyl ethylamine were filtered through two columns of activated basic alumina and transferred under an atmosphere of argon gas in a solvent purification system designed and manufactured by J. C. Meyer (University of California, Irvine). Anhydrous DMF was obtained by passing through two columns of activated molecular sieves. All other anhydrous solvents and reagents were purchased from Aldrich, Sigma-Aldrich, Fluka, or Acros and were used without any further purification unless otherwise stated. Final compounds were judged to be >95% pure by IH NMR analysis and confirmed by LC/MS and HPLC. LC/MS was performed on a Waters AllianceHT LC/MS with a gradient of 0-100% methanol (0.05% TFA) over 7 min. HPLC was performed on a Waters AllianceHT LC with a gradient of 0-100% acetonitrile (0.05% TFA) over 7 min.

[0138] General Procedure for t-Boc Deprotection. The protected amine (31.2 mg, 0.054 mmol) was dissolved in a 1 or 3 N HCl solution in ethyl acetate (2 mL, anhydrous), and the reaction mixture was stirred at ambient temperature for 5-15 h. A white precipitate formed after several minutes. Additional HCl was added as needed (2 mL), and the reaction mixture was stirred until complete by TLC. The reaction was completed as described below.

[0139] General Procedure for SiIyI Deprotection. To a stirred solution of the protected phenol (1.0 mmol) in THF (10 mL) was added TBAF (1.5 mL, 1.5 mmol, 1 M solution in THF) dropwise. The reaction mixture was stirred for 10-30 min until complete by TLC analysis and then diluted with ethyl acetate. The reaction mixture was washed with 0.5 M HCl, and the aqueous phase was extracted with ethyl acetate. The combined organic layers were sequentially washed with water and brine and then dried over MgSO4. The crude product was purified as described below.

[0140] Thyronamine Hydrochloride (TOAM). Refer to the general procedure for t-Boc deprotection described above. The crude reaction mixture was concentrated in vacuo and dried under high vacuum pressure to give 23 as a slightly tan solid (32.9 mg, 100% yield). IH NMR (400 MHz, DMSO-cfό): δ 9.37 (s, 1 H), 7.90 (br s, 3 H), 7.20 (d, J) 8.4 Hz, 1 H), 6.86 (ovrlp d, J) 8.8 Hz, 1 H), 6.85 (ovrlp d, J) 8.4 Hz, 1 H), 6.78 (d, J) 8.8 Hz, 1 H), 2.99 (app br q, J) 8.0 Hz, 2 H), 2.81 (t, J) 8.2 Hz, 2 H). HRMS (EI+, free base) mlz for C14H15NO2: calcd, 229.1103; found, 229.1107. LC/MS (LC: gradient 0-100% MeOH [0.05% TFA], MS: EI+): retention time, 4.78 min; purity, 100%; [M - NH2]+ calcd, 213.09; found, 213.37 mlz. HPLC (gradient 0-100% MeCN [0.05% TFA]): retention time, 3.22 min; purity, 95%.

[0141] 3-Iodothyronamine Hydrochloride (TlAM). Refer to the general procedure for t- Boc deprotection described above. The crude precipitate was filtered and washed with ether to give 1 as a white solid (816 mg, 93% yield). IHNMR (400 MHz, DMSO-^6): δ 9.44 (s, 1 H), 8.12 (br s, 3 H), 7.76 (s, 1 H), 7.20 (d, J) 8.0 Hz, 1 H), 6.79 (s, 4 H), 6.68 (d, J) 8.4 Hz, 1 H), 2.98 (app br q, J) 7.2 Hz, 2 H), 2.84 (t, J) 7.4 Hz, 2 H). HRMS (EI+, free base) mlz for C14H14INO2 [M - NH3]+: calcd, 337.9804; found, 337.9812. LC/MS (LC: gradient 0-100% MeOH [0.05% TFA], MS: EI+): retention time, 5.32 min; purity, 100%; [M + H]+: calcd, 356.01; found, 356.44 mlz, HPLC (gradient 0-100% MeCN [0.05% TFA]): retention time, 3.73 min; purity, 100%.

[0142] 3'-Iodothyronamine Hydrochloride (3'-TlAM). Refer to the general procedure for t-Boc deprotection described above. The crude reaction mixture was concentrated in vacuo and dried under high vacuum pressure to give 25 as a white solid (12.7 mg, 98% yield). IH NMR (400 MHz, DMSO-c/6): δ 10.24 (s, 1 H), 7.86 (s, 3 H), 7.30 (d, J) 2.4 Hz, 1 H), 7.23 (d, J) 8.4 Hz, 2 H), 6.96-6.86 (m, 4 H), 3.01 (br s, 2 H), 2.83 (app t, J) 7.6 Hz, 2 H). HRMS (EI+, free base) mlz for C14H14INO2 [M - NH3]+: calcd, 337.9804; found, 337.9809. LC/MS (LC: gradient 0-100% MeOH [0.05% TFA], MS: EI+): retention time, 5.48 min; purity, 100%; [M + H]+: calcd, 356.01; found, 356.44 mlz. HPLC (gradient 0-100% MeCN [0.05% TFA]): retention time, 3.84 min; purity, 100%.

[0143] 0-(p-Fluoro)phenyl-3-iodotyramine Hydrochloride: Refer to the general procedure for t-Boc deprotection above. The reaction was concentrated to dryness and dried in vacuo to give 92 as a slightly yellow residue (43.6 mg, 97% yield). IH NMR (400 MHz, DMSO-^6): δ 8.72 (br s, 2 H), 7.79 (d, J) 0.8 Hz, 1 H), 7.25 (dd, J) 8.4, 1.2 Hz, 1 H), 7.17 (app t, J) 8.8 Hz, 2 H), 6.94-6.87 (m, 3 H), 3.08 (app t, J) 8.0 Hz, 2 H), 2.86 (app t, J) 7.8 Hz, 2 H), 2.51 (s, 3 H). HRMS (EI+, free base) mlz for C15H15-FICO: calcd, 371.0182; found, 371.0178. LC/MS (LC: gradient 0-100% MeOH [0.05% TFA], MS: EI+): retention time, 5.87 min; purity, 100%; [M + H]+: calcd, 372.03; found, 372.50 mlz. HPLC (gradient 0-100% MeCN [0.05% TFA]): retention time, 4.37 min; purity, 98%.

[0144] In view of the many possible embodiments to which the principles of the disclosed invention may be applied, it should be recognized that the illustrated embodiments are only preferred examples of the invention and should not be taken as limiting the scope of the invention. Rather, the scope of the invention is defined by the following claims. We therefore claim as our invention all that comes within the scope and spirit of these claims.

Claims

We claim:

1. A method of cytoprotection comprising administering to the subject an effective amount of a thyronamine preconditioning agent prior to an event that produces cellular injury, thereby protecting the cell against injury.

2. The method of claim 1, wherein the method comprises a method of protecting a subject against injury caused by an excitotoxic, ischemic and/or hypoxic event, and the method comprises selecting a subject that is at risk of an excitotoxic, ischemic and/or hypoxic event and administering the effective amount of the thyronamine preconditioning agent sufficiently in advance of the event to protect the cell against injury.

3. The method of claim 1, wherein the thyronamine preconditioning agent comprises:

wherein X represents -CR₂-, -C(O)CH₂-, -(CH₂)_m-; each R independently is selected from H and lower alkyl; m is from 0 to 4; n is from 1 to 6; Y is -NR¹R², or OR³;

R¹ and R² independently are H, lower alkyl, benzyl, or together form a cycloalkyl group; R³ is H, lower alkyl or acyl;

R⁴ and R⁵ independently are H, cyano, halo, lower alkyl, fluoroalkyl, or -OR¹¹; R¹¹ is H, acyl or lower alkyl;

R⁶, R⁷, R⁸, R⁹ and R¹⁰ independently are selected from H, halogen, lower alkyl, haloalkyl nitro, or -OR¹²; and

R¹² is H, acyl or lower alkyl.

4. The method of claim 3, wherein the thyronamine preconditioning agent comprises:

wherein at least one of R⁴, R⁵, R⁶ and R¹⁰ is an iodo group.

5. The method of claim 4, wherein at least one of R⁴ and/or R⁵ is iodo.

6. The method of claim 4, wherein at least one of R⁴-R¹⁰ is acyloxy (-OC(O)R), alkoxy, or hydroxy.

7. The method of claim 3, wherein the thyronamine preconditioning agent is

or

wherein wherein X represents -CR₂-, -C(O)CH₂-, -(CH₂)_m-; each R independently is selected from H and lower alkyl; m is from 0 to 4; n is from 1 to 6;

Y is -NR¹R², or OR³;

R¹ and R² independently are H, lower alkyl, benzyl, or together form a cycloalkyl group;

R³ is H, lower alkyl or acyl; R⁴ and R⁵ independently are H, cyano, halo, lower alkyl, fluoroalkyl, or -OR¹¹; R¹¹ is H, acyl or lower alkyl;

R⁶, R⁷, R⁸, R⁹ and R¹⁰ independently are selected from H, halogen, lower alkyl, haloalkyl nitro, or -OR¹²; and

R¹² is H, acyl or lower alkyl.

8. The method of claim 2, wherein selecting the subject at risk for an excitotoxic, ischemic and/or hypoxic event comprises selecting a subject at risk of neurological injury.

9. The method of claim 8, wherein selecting the subject comprises selecting a subject who has atrial fibrillation, or who has been determined to have had one or more transient ischemic events, a stroke, and/or hypertension.

10. The method of claim 8, wherein selecting the subject comprises selecting a subject who is to undergo a surgical procedure that carries a risk of ischemic injury.

11. The method of claim 10, wherein the surgical procedure is a vascular surgical procedure.

12. The method of claim 11, wherein the surgical procedure is an endarterectomy, a pulmonary bypass or a coronary artery bypass surgery.

13. The method of claim 8, wherein selecting the subject comprises selecting a subject who is at risk because of participation in a contact sport or combat.

14. The method of claim 1, wherein administering the composition comprises administering the thyronamine preconditioning agent at least about 10 hours prior to the event.

15. The method of claim 1, wherein administering the thyronamine preconditioning agent comprises administering a plurality of doses of the composition, wherein the final dose is administered within 1 week prior to the event.

16. The method of any one of claims 1-7, wherein the cell is a neural cell, a muscle cell, a liver cell, a kidney cell, an endothelial cell or an immune system cell .

17. The method of claim 16, wherein the neural cell is a hippocampal neuron or a cortical neuron.

18. The method of claim 5, wherein the muscle cell is a cardiac muscle cell.

19. The method of any one of claims 1-18, wherein the subject is human.

20. The method of any one of claims 1-18, wherein the hypoxia is associated with hypoxia in utero or an ischemic event.

21. The method of any one of claims 1-7, wherein the event comprises a stroke.

22. The method of any one of claims 1-7, wherein the event is traumatic brain injury.

23. The method of any one of claims 1-7, wherein administering the composition comprising the thyronamine preconditioning agent results in hypothermia that induces a cytoprotective effect.

24. The method of any one of claims 1-23, comprising administering the composition comprising the thyronamine preconditioning agent to a subject transdermally, orally, intranasally, intrathecally, intravenously or intraperitoneally.

25. The method of any one of claims 1-24, wherein the thyronamine preconditioning agent is 3-iodothyronamine, 3-methylthyronamine, N-methyl-O-(p-trifluoromethyl)benzyl- tyramine hydrochloride, O-phenyl-3-iodotyramine hydrochloride or 0-(p-Fluoro)phenyl-3- iodotyramine hydrochloride.

26. The method of any one of claims 1-25, wherein the thyronamine preconditioning agent is 3-iodothyronamine (T₁AM).

27. The method of any one of claims 1-25, wherein the thyronamine preconditioning agent induces hypothermia to an extent equal to or greater than TlAM.

28. The method of any one of claims 1-27, comprising administering a preconditioning dose of the thyronamine preconditioning agent that is at least about 0.005 mg/kg and no more than about 0.5 mg/kg.

29. The method of claim 28, comprising administering a preconditioning dose of the thyronamine preconditioning agent of at least about 0.02 mg/kg and no more than about 0.2 mg/kg.

30. Use of an effective amount of a thyronamine preconditioning agent in the preparation of a medicament for the prophylactic treatment of a cellular injury before the injury occurs, wherein the agent is administered in advance of the cellular injury.

31. Use of the composition of claim 30, wherein the cellular injury results from an excitotoxic, ischemic and/or hypoxic event.

32. Use of the composition of claim 31, wherein the cellular injury results from an hypoxic event.

33. Use of the composition of claim 32, wherein the hypoxic event results from ischemia.

34. Use of the composition of claim 30, wherein the cellular injury is a neurological injury.

35. Use of the composition of claim 34, wherein the neurological injury is a stroke.

36. Use of the composition of claim 30, wherein the thyronamine preconditioning agent comprises

wherein X represents -CR₂-, -C(O)CH₂-, -(CH₂),,,-; each R independently is selected from H and lower alkyl; m is from 0 to 4; n is from 1 to 6; Y is -NR¹R², or OR³;

R¹ and R² independently are H, lower alkyl, benzyl, or together form a cycloalkyl group; R³ is H, lower alkyl or acyl;

R⁴ and R⁵ independently are H, cyano, halo, lower alkyl, fluoroalkyl, or -OR¹¹; R¹¹ is H, acyl or lower alkyl;

R⁶, R⁷, R⁸, R⁹ and R¹⁰ independently are selected from H, halogen, lower alkyl, haloalkyl nitro, or -OR¹²; and

R¹² is H, acyl or lower alkyl.

37. Use of the composition of claim 36, wherein the agent comprises

wherein at least one of R⁴, R⁵, R⁶ and R¹⁰ is an iodo group.

38. Use of the composition of claim 37, wherein at least one of R⁴ and/or R⁵ is iodo.

39. Use of the composition of claim 37, wherein at least one of R⁴-R¹⁰ is acyloxy (- OC(O)R), alkoxy, or hydroxy.

40. Use of the composition of claim 36, wherein the thyronamine preconditioning agent comprises

or

wherein X represents -CR₂- -C(O)CH₂-, -(CH₂)_m-; each R independently is selected from H and lower alkyl; m is from 0 to 4; n is from 1 to 6; Y is -NR¹R², or OR³;

R¹ and R² independently are H, lower alkyl, benzyl, or together form a cycloalkyl group; R³ is H, lower alkyl or acyl;

R⁴ and R⁵ independently are H, cyano, halo, lower alkyl, fluoroalkyl, or -OR¹¹; R¹¹ is H, acyl or lower alkyl;

R⁶, R⁷, R⁸, R⁹ and R¹⁰ independently are selected from H, halogen, lower alkyl, haloalkyl nitro, or -OR¹²; and

R¹² is H, acyl or lower alkyl.

41. Use of the composition of claim 36, wherein the thyronamine preconditioning agent is 3-iodothyronamine, 3-methylthyronamine, N-methyl-O-(p-trifluoromethyl)benzyl-tyramine hydrochloride, O-phenyl-3-iodotyramine hydrochloride or (9-(p-Fluoro)phenyl-3- iodotyramine hydrochloride.

42. Use of the composition of claim 41, wherein the thyronamine preconditioning agent is 3-iodothyronamine (TiAM).

43. Use of the composition of claim 30, wherein the agent is administered 10 hours to two days in advance of the injury.