CN100574758C - Compounds for the treatment of ischemia-related diseases - Google Patents

Abstract

The present invention relates to treat the method for ischemic related conditions for the patient who needs by using the thiosemicarbazones chemical compound.The preferred embodiment of the invention relates to the method for the treatment of specific ischemic related conditions, described disease includes but not limited to alzheimer disease, parkinson, CC is built bridge and is performed the operation, the diffusivity cerebral ischemia that asystole causes, the focus cerebral infarction, cerebral hemorrhage, red infarct, hypertensive cerebral is hemorrhage, the cerebral hemorrhage that breaks and cause by intracranial vascular malformation, the subarachnoid hemorrhage that rupture of intracranial aneurysm causes, hypertensive encephalopathy, the cerebral ischemia that carotid artery stenosis or obstruction cause, heart source property thromboembolism, spinal cord apoplexy and spinal cord injury, cerebrovascular disease: for example, atherosclerosis, vasculitis, the speckle degeneration, myocardial infarction, myocardial ischemia and supraventricular tachycardia.

Description

Compounds for the treatment of ischemia-related diseases

field of invention

The present invention relates to methods of treating ischemia-related diseases and disorders, including neurological and cardiac diseases due to sudden loss of oxygen, and degenerative diseases such as Alzheimer's disease.

Background technique

The central nervous system (CNS) consists of the spinal cord, brain and retina, and contains hundreds of billions of nerve cells (neurons), thus forming a network capable of complex functions. The existence of CNS neurons and the realization of physiological functions require energy support. The only source of energy for central nervous cells is generally considered to be glucose and oxygen from the blood. If the blood supply to all or any part of the central nervous system is shut off, the affected neurons become deprived of oxygen and glucose (also known as ischemia) and rapidly degenerate. Clinically, the lack of blood supply to the nervous system is usually called "stroke". A mere interruption of oxygen supply is called "hypoxia," which occurs during fainting, suffocation, or drowning. The interruption of glucose supply alone is called "low sugar", such as occurs in diabetic patients who take too much insulin. All of the above conditions, involving energy deficits, are considered clinically as underlying causes of brain damage. Hereinafter, "energy deficit" and "ischemia" are used interchangeably, but both mean any condition that starves the central nervous system of the energy it needs.

In the past few years, neuroscientists have made great strides in understanding the mechanisms of neurodegeneration caused by energy shortages. It has been recognized that glutamate is an important excitatory neurotransmitter in the normal and healthy condition of the central nervous system, but plays a neurotoxic role in ischemia, called "excitotoxicity". Normally, glutamate exists in the cell and is only released in small amounts at synaptic junctions, acting on adjacent neurons containing glutamate receptors to transmit nerve signals. Under normal conditions, glutamate released into the extracellular fluid is transported back into the neuron in a highly efficient transport process within milliseconds.

As long as this transport process works properly, the excitotoxic potential of glutamate is curbed. However, this transport process requires energy, so in the case of ischemia (energy shortage), glutamate transport fails, and glutamate molecules released as transmitters will be released at extracellular synapses. accumulate in the liquid. This keeps glutamate in constant contact with excitatory receptors, putting those receptors in an overstimulated state, in which case neurons become overexcited and die. Two additional factors complicate and exacerbate the situation: (1) overstimulated neurons release excess glutamate at additional synaptic connections, leaving more neurons in an overstimulated state and neurotoxic accumulation range beyond the initial ischemic zone; and (2) overstimulated neurons consume glucose or oxygen more rapidly than normal neurons, which accelerates the consumption of limited energy sources and further impairs glutamate transport process. Thus, brain damage in situations of energy deprivation such as stroke, cardiac arrest, fainting hypoxia, or low glucose is caused by a complex mechanism; the initial cause is ischemia itself, but the resulting glutamine Malfunction of the salt transport system and the glutamate-mediated cascade of excitotoxic events are largely responsible for subsequent brain injury.

In addition to the above, it has recently been noted that various defects in the ability of neurons to use energy substrates (glucose and oxygen) to maintain their energy levels can also stimulate excitotoxic processes leading to neuronal death. This is postulated to be the mechanism of neurodegeneration that occurs in neurological diseases such as Alzheimer's disease, Parkinson's disease, Huntington's disease and amyotrophic lateral sclerosis. For example, evidence of defects in intracellular energy metabolism has been found in brain biopsies of patients with Alzheimer's disease and has been suggested to trigger the release of the excitotoxic potential of glutamate leading to neuronal death in Alzheimer's disease, thereby Explained by an excitotoxic process associated with energy. Evidence for intrinsic defects in energy metabolism in cells has also been reported in Parkinson's disease and Huntington's disease. Therefore, rational therapeutic strategies to prevent neurodegeneration in these diseases would include approaches to correct energetic deficits or prevent excitotoxic neurodegeneration.

Neurodegenerative diseases are a group of diseases characterized by changes in normal neuronal function, in most cases leading to neuronal death (most of these diseases are associated with severe neurological damage, especially in advanced stages). In most cases, the cause is unknown and the disease is progressive. Neurodegenerative diseases invariably result in severe emotional, physical, and financial stress, affecting not only individuals but also social burdens.

The most consistent risk factor for developing a neurodegenerative disorder, particularly Alzheimer's disease or Parkinson's disease, is advancing age. For a century, in industrialized countries, the growth rate of the population aged 65 and over has far exceeded the growth rate of the population as a whole. Thus, it can be expected that within a few generations the proportion of the population that is elderly will double; and with it, the proportion of people at risk of developing neurodegenerative diseases will also double. Such predictions are at the center of heightened concern among the medical profession and lawmakers, as the emotional, physical and financial burdens on patients, caregivers and society associated with disabling diseases can easily be expected to be large. increase in magnitude. In fact, to address this problem, there are several approved drugs that can alleviate the symptoms of several neurodegenerative diseases to some extent. The long-term use of these drugs often has some degree of side effects, and none of them seem to be able to stop. Transgender process. Relatedly, our limited understanding of the causes and mechanisms of neuronal death in neurodegenerative diseases hinders the development of effective preventive or protective treatments. Despite the bleak outlook, several neurobiological breakthroughs have brought us closer than ever to unraveling the secrets of several neurodegenerative diseases and obtaining effective therapeutic strategies.

Significant progress has been made in developing methods to protect and reduce neuronal damage associated with CNS ischemia. The most active research in this field involves the use of receptor-specific antagonist drugs (pharmacological term for a drug that occupies and blocks a certain receptor on the cell surface without triggering the activity of the receptor, called the receptor A method for inhibiting excitatory activity at glutamate receptors. Glutamate receptors that can mediate excitotoxic neurodegeneration can be broadly divided into two categories: NMDA receptors and non-NMDA receptors. NMDA receptors are named after N-methyl-D-aspartate. This class of drugs does not exist in the brain under natural conditions, but it was found to bind strongly to glutamate receptors, so it is related to The receptors it binds to are called NMDA receptors. Recently, non-NMDA glutamate receptors have been divided into two different types, one is KA (kainic acid) receptors, and the other is AMPA receptors (formerly known as QUIS receptors).

NMDA receptor antagonists have been shown repeatedly to protect the CNS from ischemic neurodegeneration in vitro and in a number of in vivo animal models; The ischemia of the other major ischemia - "diffuse ischemia" may be ineffective, while providing only partial protection against the other major ischemia, "focal" ischemia. Furthermore, NMDA antagonists must be administered immediately after the onset of ischemia to confer significant protection. Experimental data on non-NMDA antagonists are more limited, but a few in vivo animal studies suggest that these antagonists confer significant protection against ischemic neurodegeneration even when administered after an ischemic event.

Although NMDA and non-NMDA antagonists alone claim to confer substantial protection against CNS ischemia, accumulating evidence suggests that their degree of protection alone is relatively limited.

A prominent limitation of glutamate receptor antagonists as neuroprotectants against ischemic neurodegeneration is that they only temporarily insulate neurons from degeneration; Other confusions come. Thus, while these antagonists did confer some degree of protection against ischemic neurodegeneration in animal models, as noted above, the protection was incomplete and in some cases only delayed the onset of degeneration . However, it is important to note that delaying the onset of degeneration may be of value if there are other drugs or approaches that provide additional and/or continued protection during this delayed period.

A critical factor that has not been adequately addressed in most ischemic studies is the timing of administration involving the damaging (ischemic) event. This is a very important consideration; although some ischemic events can be predicted (such as open heart surgery), the vast majority of ischemic events cannot be predicted, and in most cases, treatment is only performed when an ischemic event occurs started during or after the process. Because CNS cells begin to degenerate very rapidly after ischemia occurs, it is clear that new approaches to neuroprotection are needed that can be administered after CNS degeneration begins.

Another important consideration is whether ischemia is only transient (eg, during an episode of cardiac arrest) or persistent (eg, following thrombotic or embolic occlusion of a CNS vessel). If the ischemia is short-lived, the supply of blood carrying oxygen and glucose to the CNS can be restored immediately after the event, and drugs that prevent neurodegeneration or promote recovery from ischemic damage can reach the ischemic tissue through blood circulation.

If the blood supply to the brain area is permanently blocked by a blood clot, it is impossible for existing methods to prevent neurodegeneration in the center of the ischemic area, because the ischemic tissue is permanently deprived of oxygen and glucose, and drugs cannot reach the ischemic vessel through the blocked blood vessel organize. However, there is a large tissue region around the edge of the ischemic region, called the penumbra, where blood is available to adjacent CNS regions, and this tissue region is a potential target for drug therapy. Likewise, clot-dissolving drugs (thrombolytics, such as streptokinase and tissue plasminogen activator) are now being tested and developed for the treatment of heart attacks and to restore blood supply to the CNS after stroke. When these drugs are widely used in the CNS of the human body, the blood vessels can be recanalized by using these drugs, so that the ischemic CNS tissue can not only obtain oxygen and glucose, but also obtain the neurodegeneration protection disclosed here or promote recovery after ischemic damage Drug.

Finally, in exceptional cases, such as blockage of a blood clot in a major artery supplying blood to the retina of the eye, the drugs disclosed herein may be helpful. When such blood vessels are blocked, it is possible to directly inject the medicament of the present invention into the vitreous humor of the eye (ie, the clear fluid inside the eyeball) to administer to the ischemic retina. Drugs can rapidly diffuse from the vitreous into the retina.

The development of therapeutics capable of preventing or treating the disease/disorder consequences, whether acute or chronic, of ischemic events would be highly desirable.

Summary of the invention

The present invention relates to a method for treating ischemia-related diseases by administering a compound of general formula I or a prodrug thereof to a patient in need:

通式I

Formula I

in:

E is oxygen, sulfur, NH or N-C1-6 alkyl;

R ₁ , R ₂ , R ₃ and R ₄ are independently selected from hydrogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted cycloalkyl, optionally substituted Haloalkyl, optionally substituted aryl, optionally substituted aminoalkyl, optionally substituted hydroxyalkyl, optionally substituted alkoxyalkyl, and optionally substituted alkanoyl, or

NR ₁ R ₂ taken together form a 3 to 7 membered ring which may contain 0, 1 or 2 hetero ring atoms additionally selected from nitrogen, oxygen and sulfur; and

HET is an optionally substituted 5- to 7-membered heteroaryl residue containing 1 to 4 heteroatoms selected from nitrogen, oxygen, or sulfur.

A preferred embodiment relates to compositions for use in the methods of the invention wherein HET is a residue of the general formula:

in:

m is 0 or 1;

When m is 0, Z ₁ , Z ₂ and Z ₃ are independently selected from N, O, S or CR, or

When m is 1, Z ₁ , Z ₂ and Z ₃ are independently selected from N or CR;

Each occurrence of R is independently selected from hydrogen, halide, hydroxy, thiol, amino, hydroxyamino, mono-C _1-8 alkylamino, di(C _1-8 alkyl)amino, C _{1 -8} alkoxy, C _1-8 alkyl, C _2-8 alkenyl and C _2-8 alkynyl; and

x is an integer from 0 to 4.

More preferred embodiments include the use of compositions wherein the HET residues are independently selected from pyridyl, pyrazinyl, pyrimidinyl, pyrrolyl, imidazolyl, triazolyl, oxazolyl and thiaxazolyl; and

R ₁ , R ₂ , R ₃ and R ₄ are independently selected from hydrogen, C _1-8 alkyl, C _2-8 alkenyl, C _2-8 alkynyl, C _3-8 cycloalkyl, C _1-8 Haloalkyl, C _6-10 aryl, amino C 1-8 alkyl _, hydroxy C _1-8 alkyl, C _1-8 alkoxy C _1-8 alkyl and C _1-8 alkanoyl, or

NR ₁ R ₂ taken together form a 3 to 7 membered ring which may contain 0, 1 or 2 additional hetero ring atoms selected from nitrogen, oxygen and sulfur.

Another embodiment of the present invention relates to a method of treating ischemia-related diseases by administering to a patient in need thereof a compound of the following general formula II or a prodrug thereof:

通式II

Formula II

in:

R, R ₁ , R ₂ , R ₃ and R ₄ are independently selected from hydrogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted cycloalkyl, optionally Substituted haloalkyl, optionally substituted aryl, optionally substituted aminoalkyl, optionally substituted hydroxyalkyl, optionally substituted alkoxyalkyl, and optionally substituted alkanoyl, or

NR ₁ R ₂ combine to form a 3 to 7 membered ring which may contain 0, 1 or 2 additional hetero ring atoms selected from nitrogen, oxygen and sulfur, and

x is an integer from 0 to 5.

And, more specifically here:

x is 0, 1, 2 or 3

R, R ₁ , R ₂ , R ₃ and R ₄ are independently selected from hydrogen, C _1-8 alkyl, C _2-8 alkenyl, C _2-8 alkynyl, C _3-8 cycloalkyl, C _{1 -8} haloalkyl, C _6-10 aryl, amino C 1-8 alkyl, hydroxy C _1-8 alkyl _{, C 1-8 alkoxy C 1-8 alkyl and C 1-8 alkanoyl , or}

NR ₁ R ₂ taken together form a 3 to 7 membered ring which may contain 0, 1 or 2 additional hetero ring atoms selected from nitrogen, oxygen and sulfur.

Another embodiment of the present invention relates to a method of treating ischemia-related diseases by administering to a patient in need thereof a compound of the following general formula III or a prodrug thereof:

通式III

Formula III

in:

R, R ₁ , R ₂ , R ₃ and R ₄ are independently selected from hydrogen, C _1-8 alkyl, C _2-8 alkenyl, C _2-8 alkynyl, C _3-8 cycloalkyl, C _{1 -8} haloalkyl, C _6-10 aryl, amino C 1-8 alkyl, hydroxy C _1-8 alkyl _{, C 1-8 alkoxy C 1-8 alkyl and C 1-8 alkanoyl , or}

NR ₁ R ₂ taken together form a 3 to 7 membered ring which may contain 0, 1 or 2 additional hetero ring atoms selected from nitrogen, oxygen and sulfur;

R ₆ is hydrogen, hydroxyl, amino or C _1-8 alkyl;

R _{and R} are independently selected from hydrogen, halide, hydroxyl, mercapto, amino, hydroxylamino, mono-C _1-8 alkylamino, di(C _1-8 alkyl)amino, C _1-8 alkoxy group, C _1-8 alkyl, C _2-8 alkenyl and C _2-8 alkynyl.

The most preferred embodiment of the present invention relates to a method of treating ischemia-related diseases by administering PAN811 (3-aminopyridine-2-carboxyaldehyde thiosemicarbazone) having the following general formula to a patient in need thereof:

Preferred embodiments of the invention relate to methods of treating certain ischemia-related diseases including, but not limited to, Alzheimer's disease, Parkinson's disease, coronary artery bypass surgery, diffuse cerebral infarction due to cardiac arrest Ischemia, focal cerebral infarction, cerebral hemorrhage, hemorrhagic infarction, hypertensive hemorrhage, cerebral hemorrhage caused by abnormal rupture of intracranial blood vessels, subarachnoid hemorrhage caused by ruptured intracranial aneurysm, hypertensive encephalopathy, cervical Cerebral ischemia due to arterial stenosis or occlusion, cardiogenic thromboembolism, spinal cord stroke and spinal cord injury, cerebrovascular disease: eg, atherosclerosis, vasculitis, plaque degeneration, myocardial infarction, myocardial ischemia, and supraventricular Tachycardia.

Brief description of the drawings

Figure 1. Survival of cells pretreated with PAN-811 (A) or known neuroprotective agents vitamin E (B), lipoic acid (C), or Ginkgo Biloba (D) followed by hydrogen peroxide treatment Representative graphs of (left panel) and neuroprotective capacity (right panel).

Figure 2 is a representative illustration of the effect of PAN-811 on ROS production in neural cells. (A) Effect of PAN-811 on the production of hydrogen peroxide-induced ROS in neuronal cells. (B) Effect of PAN-811 on the production of basal levels of ROS in neuronal cells.

Figure 3 is a representative graphical representation of the dependence of neurotoxicity on glucose concentration under hypoxic conditions.

Figure 4 shows representative histological images of cells with and without the neuroprotectants MK-801 and PAN-811 under hypoxia.

Figure 5 is a representative graph of the neuroprotective effect of PAN-811 under normoxia and hypoxia.

Figure 6 is a representative graphic representation of PAN-811 toxicity under hypoxic/low glucose conditions.

Figure 7 is a representative illustration of the protective effect of PAN-811 on neuronal cell death under mild hypoxia/low glucose conditions.

Figure 8 is a representative graph of PAN-811 neurotoxicity when cerebral cortical neurons were treated with PAN-811 for 24 hours.

Figure 9 is a representative graphical representation of the protective effect of PAN-811 against ischemic toxicity.

Figure 10 is a representative graph of cell viability following pretreatment with PAN-811 or solvent, followed by hydrogen peroxide treatment.

Figure 11 is a representative graph of cell viability following pretreatment with PAN-811 or solvent, followed by treatment with hydrogen peroxide.

Detailed description of the invention

Features and other details of the invention will now be more particularly described and pointed out in the claims. It should be understood that the particular embodiments of the invention are shown by way of illustration and not limitation of the invention. The principal features of this invention can be employed in different embodiments without departing from the scope of the invention.

The pathology of an ischemia-related disorder or disease involving reduced blood supply to an internal organ, tissue, or part of the body, usually as a result of narrowing or blockage of blood vessels, such as retinopathy, acute renal failure, myocardial infarction, and stroke. This can be the result of an acute episode (such as a heart attack) or a chronic process (such as a neurodegenerative disease).

The present invention relates to a method for administering a compound of general formula I or a prodrug thereof to a patient in need to treat ischemia-related diseases:

通式I

Formula I

in:

E is oxygen, sulfur, NH or N-C1-6 alkyl;

R ₁ , R ₂ , R ₃ and R ₄ are independently selected from hydrogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted cycloalkyl, optionally substituted Haloalkyl, optionally substituted aryl, optionally substituted aminoalkyl, optionally substituted hydroxyalkyl, optionally substituted alkoxyalkyl, and optionally substituted alkanoyl, or

NR ₁ R ₂ taken together form a 3 to 7 membered ring which may contain 0, 1 or 2 additional hetero ring atoms selected from nitrogen, oxygen and sulfur; and

HET is an optionally available 5- to 7-membered heteroaryl residue containing 1 to 4 heteroatoms selected from nitrogen, oxygen, or sulfur.

A preferred embodiment relates to compositions for use in the methods of the invention wherein HET is a residue of the general formula:

in:

m is 0 or 1;

When m is 0, Z ₁ , Z ₂ and Z ₃ are independently selected from N, O, S or CR or

When m is 1, Z ₁ , Z ₂ and Z ₃ are independently selected from N or CR;

Each occurrence of R is independently selected from hydrogen, halide, hydroxy, mercapto, amino, hydroxyamino, mono-C _1-8 alkylamino, di(C _1-8 alkyl)amino, C _1-8 alkane Oxygen, C _1-8 alkyl, C _2-8 alkenyl and C _2-8 alkynyl; and

x is an integer from 0 to 4.

More preferred embodiments include the use of compositions wherein the HET residues are independently selected from pyridyl, pyrazinyl, pyrimidinyl, pyrrolyl, imidazolyl, triazolyl, oxazolyl and thiaxazolyl; and

R ₁ , R ₂ , R ₃ and R ₄ are independently selected from hydrogen, C _1-8 alkyl, C _2-8 alkenyl, C _2-8 alkynyl, C _3-8 cycloalkyl, C _1-8 Haloalkyl, C _6-10 aryl, amino C 1-8 alkyl _, hydroxy C _1-8 alkyl, C _1-8 alkoxy C _1-8 alkyl and C _1-8 alkanoyl, or

NR ₁ R ₂ taken together form a 3 to 7 membered ring which may contain 0, 1 or 2 additional hetero ring atoms selected from nitrogen, oxygen and sulfur.

Another embodiment of the present invention relates to a method of treating ischemia-related diseases by administering a compound of general formula II or a prodrug thereof to a patient in need thereof:

通式II

Formula II

in:

R, R ₁ , R ₂ , R ₃ and R ₄ are independently selected from hydrogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted cycloalkyl, optionally Substituted haloalkyl, optionally substituted aryl, optionally substituted aminoalkyl, optionally substituted hydroxyalkyl, optionally substituted alkoxyalkyl, and optionally substituted alkanoyl, or

NR ₁ R ₂ taken together form a 3 to 7 membered ring which may contain 0, 1 or 2 additional hetero ring atoms selected from nitrogen, oxygen and sulfur, and

x is an integer from 0 to 5.

And, more specifically among them:

x is 0, 1, 2 or 3,

R, R ₁ , R ₂ , R ₃ and R ₄ are independently selected from hydrogen, C _1-8 alkyl, C _2-8 alkenyl, C _2-8 alkynyl, C _3-8 cycloalkyl, C _{1 -8} haloalkyl, C _6-10 aryl, amino C 1-8 alkyl _, hydroxy C _1-8 alkyl, C _1-8 alkoxy C _1-8 alkyl and C _1-8 alkanoyl, or

NR ₁ R ₂ taken together form a 3 to 7 membered ring which may contain 0, 1 or 2 additional hetero ring atoms selected from nitrogen, oxygen and sulfur.

Another embodiment of the present invention relates to a method of treating ischemia-related diseases by administering a compound of formula III or a prodrug thereof to a patient in need thereof:

通式III

Formula III

in:

R, R ₁ , R ₂ , R ₃ and R ₄ are independently selected from hydrogen, C _1-8 alkyl, C _2-8 alkenyl, C _2-8 alkynyl, C _3-8 cycloalkyl, C _1-8 haloalkyl, C _6-10 aryl, amino C _{1-8 alkyl, hydroxy C 1-8 alkyl} , C _1-8 alkoxy C _1-8 alkyl and C _1-8 alkanoyl, or

NR ₁ R ₂ taken together form a 3 to 7 membered ring which may contain 0, 1 or 2 additional hetero ring atoms selected from nitrogen, oxygen and sulfur;

R ₆ is hydrogen, hydroxyl, amino or C _1-8 alkyl;

R ₅ and R ₇ are selected from hydrogen, halide, hydroxyl, mercapto, amino, hydroxyl amino, mono-C _1-8 alkylamino, two (C _1-8 alkyl) amino, C _1-8 alkoxy, C _1-8 alkyl, C _2-8 alkenyl and C _2-8 alkynyl.

The most preferred embodiment of the present invention relates to a method of treating ischemia-related diseases by administering PAN811 (3-aminopyridine-2-carboxyaldehyde thiosemicarbazone) having the following general formula to a patient in need thereof:

Preferred embodiments of the present invention relate to methods of treating certain ischemia-related diseases including, but not limited to, Alzheimer's disease, Parkinson's disease, coronary artery bypass surgery, diffuse cerebral infarction due to cardiac arrest Ischemia, focal cerebral infarction, cerebral hemorrhage, hemorrhagic infarction, hypertensive hemorrhage, cerebral hemorrhage caused by abnormal rupture of intracranial blood vessels, subarachnoid hemorrhage caused by ruptured intracranial aneurysm, hypertensive encephalopathy, cervical Cerebral ischemia due to arterial stenosis or occlusion, cardiogenic thromboembolism, spinal cord stroke and spinal cord injury, cerebrovascular disease: e.g., atherosclerosis, vasculitis, plaque degeneration, myocardial infarction, myocardial ischemia, and supraventricular arrhythmia Excessive speed.

Methods for the synthesis of compounds useful in the methods of the invention are well known in the art. Such synthetic schemes are described in several US patents: 5,281,715, 5,767,134, 4,447,427, 5,869,676 and 5,721,259. They are hereby incorporated by reference in their entirety.

Pharmaceutical Compositions Another aspect of the present invention relates to pharmaceutical compositions of thiosemicarbazones for use in the methods of the present invention. The pharmaceutical compositions of the invention generally comprise a compound for use in the methods of the invention and a pharmaceutically acceptable carrier. As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. The type of carrier can be selected according to the intended route of administration. In various embodiments, the carrier is suitable for intravenous, intraperitoneal, subcutaneous, intramuscular, topical, transdermal or oral administration. Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders suitable for the extemporaneous preparation of sterile injectable solutions or dispersions. The use of such media and agents as pharmaceutically active substances is well known in the art. Any conventional media or reagents are contemplated for use in the pharmaceutical compounds of the present invention unless incompatible with the active compounds. Supplementary active compounds can also be incorporated into the compositions.

Pharmaceutical compositions for therapeutic use typically must be sterile and stable under the conditions of manufacture and storage. Such compositions can be formulated as solutions, microemulsions, liposomes, or other ordered structures suitable to high concentrations of drug. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (such as glycerol, propylene glycol, liquid polyethylene glycol, and the like), and other suitable mixtures thereof. For example, proper fluidity can be maintained by the use of coatings such as lecithin, maintaining the desired particle size in dispersion and the use of surfactants. In many cases it will be more preferable to include isotonic agents, such as sugars, polyols like mannitol and sorbitol, or sodium chloride in the compositions. Delayed absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example monostearate or gelatin. In addition, the compounds can be administered in time-release dosage forms, for example in compositions comprising slow-release polymers. The active compounds can be prepared with carriers providing rapid release of the compound, such as such controlled release dosage forms, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used for this, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, polylactic acid, polylactic polyglycolic acid (PLG). Many methods of preparing such dosage forms are well known in the art.

Injectable sterile solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

Depending on the route of administration, the compound may be coated with a material to protect it from enzymes, acids, and other natural conditions that may inactivate the agent. For example, the compound may be co-administered with an enzyme inhibitor in a suitable carrier or diluent or administered to a subject in a suitable carrier such as liposomes. Pharmaceutical diluents include saline and aqueous buffered solutions. Enzyme inhibitors include pancreatic trypsin inhibitor, diisopropylfluorophosphate (DEP) and aprotinin (trasylol). Liposomes include water-in-oil-in-water emulsions and conventional liposomes (Strejan, et al., (1984) J. Neuroimmunol 7:27). Dispersions can also be prepared in glycerol, liquid polyethylene glycols, mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations may contain a preservative to prevent the growth of microorganisms.

The active agent in the composition, ie, one or more thiosemicarbazones, is preferably formulated in the composition in a therapeutically effective amount. "Therapeutically effective amount" means an effective amount at dosages and for periods of time necessary to achieve the desired therapeutic result, thereby affecting the course of treatment of a particular disease state. A therapeutically effective amount of an active agent may vary depending on the individual's disease state, age, sex, body weight and the ability of the active agent to elicit a desired response in the individual. Dosage regimens may be adjusted to provide the optimum therapeutic response. A therapeutically effective amount also refers to an amount in which toxic or detrimental effects are outweighed by therapeutically beneficial effects. In another embodiment, the active agent is formulated in the composition in a prophylactically effective amount. "Prophylactically effective amount" means an effective amount to obtain the desired disease prevention results at dosages and for the required time period. Typically, a prophylactic dose is less than a therapeutically effective dose because it is administered to a subject prior to onset or early in the disease.

The amount of the active compound in the composition may vary depending on the individual's disease state, age, sex, body weight and other factors. Dosage regimens may be adjusted to provide the optimum therapeutic response. For example: administration may be a single bolus, several divided doses administered over time, or the dose may be proportionally increased or decreased as indicated by the therapeutic exigencies. It is especially advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Unit dosage form as used herein refers to physically discrete units suited as unitary dosages for the mammalian subjects to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. . The dosage unit form specifications for the present invention are dictated by and are directly dependent on (a) the unique properties of the active compound and the particular therapeutic effect desired to be obtained; (b) the technical inherentness in combining such active compound with respect to individual therapeutic sensitivity. limited.

The compounds of the present invention may be formulated into pharmaceutical compositions in which the compound is the sole active agent. Alternatively, the pharmaceutical compositions may contain additional active agents. For example, two or more compounds of the present invention may be used in combination.

The present invention is further illustrated by the following examples, which should not be construed as limiting. The contents of all references, patents and published patent applications, and drawings throughout this application are hereby incorporated by reference.

Example

Example 1

Comparison of neuroprotective efficacy of PAN-811 with other neuroprotective agents

Purpose

The aim of this study was to compare PAN-811 (3-aminopyridine-2-carboxyaldehyde thiosemicarbazone; C7H9N5S; MW=195) with known neuronal Protective agents such as vitamin E, lipoic acid and ginkgo for their neuroprotective abilities.

1. Materials and methods

1.1 Study Design

1.1.1. Cell isolation and culture

Primary cortical neurons were isolated from embryonic brains of 17-day-old rats, seeded into 96-well plates at 50,000 cells per well, and cultured in conventional neurobasal medium for 2-3 weeks. Replace the medium twice with half volume of fresh neural basal medium without antioxidants.

1.1.2. Treatment with PAN-811, other neuroprotective agents and hydrogen peroxide

PAN-811 was dissolved in ethanol at 1 mg/ml (~5 mM), and further diluted with medium to final concentrations of 0.1 μM, 1 μM and 10 μM. Other known neuroprotectants were dissolved in appropriate solvents and diluted to the indicated final concentrations. Neurons were pretreated with PAN-811, known neuroprotectants, or vehicle for 24 hours, followed by hydrogen peroxide (final concentration 150 μM)-induced oxidative stress. Controls included untreated cells (no compound and hydrogen peroxide treatment), cells treated with compound only, cells exposed to hydrogen peroxide but no compound. Untreated cells were used as controls to assess toxicity and improved survival. Each analysis was repeated three times.

1.1.3. Evaluation of cell function

After 24 hours, survival and mitochondrial function were assessed using a standard MTS assay (Promega). Follow manufacturer's operating instructions.

1.2 Materials

- Neural Basal Medium (Invitrogen)

-B27-AO (Invitrogen)

-PAN-811 (Vion Pharmaceuticals, Inc.)

- Hydrogen peroxide (Calbiochem)

- Ethanol (Sigma)

-Vitamin E (Sigma)

- Lipoic acid (Sigma)

-Ginkgo (CVS)

-MTS Assay Kit (Promega)

1.3 Equipment

- Balance (Mettler-Toledo, Inc.)

-Adjustable sampler (Finnpipette)

-Cell culture cabinet (Thermo Forma)

-Cell incubator (Thermo Forma)

- Plate reader (Bio-Rad Model 550)

2. Results

2.1 Experiment

Experiments were performed as described in "Study Design" above. PAN-811 was dissolved in ethanol at 1 mg/ml (~5 mM) and further diluted with neural basal medium to final concentrations of 0.1 μM, 1 μM and 10 μM. Lipoic acid was dissolved in ethanol at 240 mM and further diluted with neural basal medium to final concentrations of 10 μM, 25 μM and 100 μM. Vitamin E was dissolved in ethanol at 100 mM and further diluted with neural basal medium to final concentrations of 50 μM, 100 μM, 200 μM and 400 μM. Ginkgo biloba was dissolved in deionized water at 6 mg/ml, and further diluted with neural basal medium to final concentrations of 2.5 μg/ml, 5 μg/ml, 25 μg/ml and 250 μg/ml. At the end of the treatment, the medium was replaced with 100 [mu]l fresh pre-warmed neural basal medium plus B27(-AO). The plates were returned to the cell incubator (37°C, 5% _CO2 ) for one hour. Afterwards, 20 μl of MTS reagent was added to each well, and the culture plate was returned to the cell culture incubator (37° C., 5% CO ₂ ) for two hours. The absorbance of each well at 490 nm was recorded with a BioRad plate reader (model 550). Wells containing medium only served as blank controls. Each data point is the average of 3 individually analyzed wells. Untreated cells were used as a control to calculate cell viability and neuroprotection. Two-week-old primary cultures were used for this study. See Figure 1 for the results.

in conclusion

At concentrations of 1-10 μM, PAN-811 showed good neuroprotective ability, even under harsh hydrogen peroxide treatment. Vitamin E and lipoic acid have minimal neuroprotective capacity under harsh handling. Ginkgo biloba showed some degree of neuroprotection under harsh handling.

At 1-10 μM final concentration, PAN-811 showed significant neuroprotection, even under harsh hydrogen peroxide treatment. The neuroprotective effect of PAN-811 is significantly higher than that of other known neuroprotective agents, such as vitamin E, lipoic acid and ginkgo.

Example 2

The Effect of PAN-811 on the Production of Reactive Oxygen Species (ROS) in Nerve Cells

Purpose

The purpose of this study was to evaluate the ability of PAN-811 to reduce ROS production in a cellular model of Alzheimer's disease-associated oxidative stress

1. Materials and methods

1.1 Study Design

1.1.1 Isolation and culture of cells

Primary cortical neurons were isolated from embryonic brains of 17-day-old rats, seeded into 96-well plates at 50,000 cells per well, and cultured in conventional neural basal medium for 2-3 weeks. Replace the medium twice with half volume of fresh neural basal medium without antioxidants.

1.1.2. Preloading cells with CM-H ₂ DCFDA dye and treating cells with PAN-811 and hydrogen peroxide

Primary cortical neurons were washed once with HBSS buffer and washed with 10 μM 5-(and 6-)chloromethyl-2',7'-dichlorodihydrofluorescein diacetate, acetyl ester (CM-H ₂ DCFDA) Incubate to preload dye. Cells were then washed with HBSS buffer, treated with PAN-811 at final concentrations of 0.1, 1, 5 and 10 μM for one hour, and further subjected to oxidative stress induced by 300 μM hydrogen peroxide for two hours.

1.1.3. Evaluation of ROS production in nerve cells

The c-DCF fluorescence of each well at 485/520 nm (Ex/Em) was recorded with a BMG polar star plate reader and used to evaluate the generation of intracellular ROS. Dye-loaded but untreated cells were used as a control for calculating c-DCF fluorescence changes. Each analysis was repeated three times.

1.2 Materials

- Neural Basal Medium (Invitrogen)

-B27-AO (Invitrogen)

- HBSS buffer (Invitrogen)

-CM-H ₂ DCFDA (Molecular Probes)

-PAN-811 (Vion Pharmaceuticals, Inc.)

- Hydrogen peroxide (Calbiochem)

- Ethanol (Sigma)

-PEG-300 (Sigma)

1.3 Equipment

- Balance (Mettler-Toledo, Inc.)

-Adjustable sampler (Finnpipette)

-Cell culture cabinet (Thermo Forma)

-Cell incubator (Thermo Forma)

-Polarstar fluorescence plate reader (BMG)

2.0 results

2.1 Experiment

Experiments were performed as described in "Study Design" above. The c-DCF fluorescence of each well at 485/520nm (Ex/Em) was recorded with a BMG pole star plate reader. Wells containing cells but no dye served as blank controls. Each data point is the mean of three individually analyzed wells. Dye-spiked but untreated cells were used as a control for calculating changes in c-DCF fluorescence. Two-week-old primary cultures were used in this study.

3.0 discussion

CM-H ₂ DCFDA is a cell _- permeable ROS indicator that fluoresces until the acetate group is removed by intracellular esterases and oxidation occurs in the cell. It is widely used to detect the production of reactive oxygen species (ROS) in cells and in animals. Here, it was used as a tool to evaluate the effect of PAN-811 on ROS generation in neural cells following the method described in the study design. As shown in Figure 2, PAN-811 showed a good ability to reduce hydrogen peroxide-induced ROS generation and the basal level of ROS generation in nerve cells. Parallel control experiments using buffer and PGE-300/EtOH instead of PAN-811 showed no effect on intracellular ROS generation. The experiment was repeated 4 times with different batches of cells and similar results were obtained. See Figure 2 for a representative experiment.

4.0 Conclusion

PAN-811 significantly reduced basal levels of ROS generation (~50%; at 10 μM) and hydrogen peroxide-induced ROS generation (~30%; at 10 μM) in primary neural cells.

references

(1).Gibson GE, Zhang H, Xu H, Park LC, Jeitner TM.(2001). Oxidative stress increases internal calcium stores and reduces a keymitochondrial enzyme. Biochim Biophys Acta. Mar16; 1586(2): 177-89.

(2). Chignell CF, Sik RH. (2003). A photochemical study of cells loaded with 2', 7'-dichlorofuorescin: implications for the detection ofreactive oxygen species generated during UVA irradiation (loaded with 2', 7'-dichlorofluorescin Photochemical studies of cells that are primed: hints for detecting reactive oxygen species produced during UVA irradiation). Free Radic Biol Med. Apr 15; 34(8): 1029-34.

Example 3

PAN-811 is a potential neuroprotectant against hypoxia or hypoxia/hypoglycemia-induced neurotoxicity

1.0 Introduction

Reducing neuronal damage in the first few minutes after stroke is an important strategy for effective treatment. During a stroke, a thrombus emboli seals off an artery, interrupting oxygen and glucose supply to a regional brain region, resulting in the loss of neurons in the infarct's central nucleus. Cells in the central nucleus die very rapidly by necrotic mechanisms. The brain area surrounding the ischemic infarction area, the structure is still there, but the (electrical conduction) function is terminated, which is called the penumbra. The penumbra is a transient area, and its evolution into infarction is a relatively progressive phenomenon (Touzani et al., 2001). This region offers the possibility to salvage some brain function, and the therapeutic window for the penumbra is substantially longer than for the infarct. The penumbra can also be described as an area of restricted blood supply where energy metabolism remains. Thus, the penumbra is a target area for neuroprotective treatments and agents that reactivate quiescent neurons, such as hyperbaric oxygen. Therefore, the damage immediately after the central nervous system injury may not be reversible, but the aggravation of brain injury, mainly the progression of diffuse cerebral hypoxic/ischemic event chain, can be prevented by effective neuroprotective strategies. For example, administration of neuroprotective agents before and during coronary artery bypass grafting (CABG) can effectively protect against neurodegeneration caused by short-term changes in blood flow in the brain (causing a mild hypoxic/hypoglycemic state) during the procedure. Thus, compounds that can significantly protect nerves and rescue neurons after nerve damage are of great importance.

2.0 purpose

The aim of this study was to understand whether PAN-811 could protect neurotoxicity induced by hypoxia or hypoxia/low glucose (H/H) in vitro. In related work, PAN-811 has shown significant neuroprotective effects on hydrogen peroxide-treated primary neurons.

3.0 Materials and methods

3.1 Materials

- Neural Basal Medium (Invitrogen)

-B27-AO (Invitrogen)

-PAN-811 (Vion Pharmaceuticals)

- Ethanol (Sigma)

-DMSO (Sigma)

-PEG-300 (Sigma)

-MTS Assay Kit (Promega)

-LDH Analysis Kit (Sigma)

3.2 Equipment

- Balance (Mettler-Toledo, Inc.)

-Adjustable sampler (Finnpipette)

-Cell culture cabinet (Thermo Forma)

-Cell incubator (Thermo Forma)

- Plate reader (Bio-Rad Model 550)

-FYRITE Gas Analyzer (Bacharach, Inc)

-Modular Incubator Chamber-101TM(Billups-Rothenberg, Inc.)

3.3 Abbreviations

BSS = balanced salt solution

CABG = coronary artery bypass surgery

d.i.v. = days in vitro

EtOH = ethanol

H/H = hypoxia/hypoglycemia

LDH = lactate dehydrogenase

MCAO = middle cerebral artery occlusion

NB = neural basal medium

NMDA = N-methyl-D-aspartate

PEG = polyethylene glycol

3.4 Study design

3.4.1 Neuronal culture

96-well culture plates were used in this experiment. Cortical neurons were seeded at a density of 50,000 cells per well on poly-D-lysine-coated surfaces and cultured in serum-free medium (NB plus B27 supplement) to obtain cultures highly enriched for neurons . Culture neurons over 14 d.i.v. to increase the susceptibility of cells to excitatory amino acids (Jiang et al., 2001). Six replicate wells were treated as a group to facilitate analysis and quantification.

3.4.2 Induction of neurotoxicity in in vitro models

As shown in the table below, glucose concentrations in the brain typically exceed 2.2 mM. During ischemia, its concentration decreased to 0.2mM and 1.4mM in the central nucleus and penumbra, respectively. Glucose concentrations return to normal levels one to two hours after restoration of circulation (Folbergrová et al., 1995).

Table 1. Glucose concentration (mmol/kg)

To understand the effect of glucose concentration on hypoxia-induced neurotoxicity, we have tested different doses of glucose. As shown in FIG. 3 , a decrease in glucose concentration to 2.9 mM did not result in neuronal cell death compared to the normal glucose concentration of 25 mM. When the glucose concentration dropped to 0.4 mM, MTS analysis showed strong cell death.

To mimic the brain environment of stroke, we established three in vitro model systems. The extreme H/H model (0.4mM glucose) is a simulation of the environment of the infarct center nucleus; the mild H/H model (1.63mM glucose) is the simulation of the environment of the penumbra during MCAO; and the simple hypoxia model (under normal neurons) , the concentration of glucose in vitro was 25 mM) to simulate the environment of the penumbra after reperfusion, because hypoxia rather than energy failure is the main cause of possible cell death after reperfusion.

Hypoxia/hypoglycemia was achieved by reducing the concentration of glucose to 0.4 mM and 1.63 mM for extreme H/H and mild H/H, respectively. BSS (116.0 mM NaCl, 5.4 mM KCl, 0.8 mM MgSO4 7H2O, 1.0 mM NaH2PO4, 1.8 mM CaCl2 2H2O, 26.2 mM NaHCO3 and 0.01 mM glycine) or BBS containing 25 mM glucose was degassed for 5 minutes before use. The medium in the plate was replaced with BBS or BBS containing glucose under hypoxia. At the same time, the medium in the plate under normoxia was replaced by non-degassed BBS or BBS containing glucose. To cause cell hypoxia, the culture plate was placed in a sealed container (Modular Culture Chamber-101TM, Billups-Rothenberg, Inc.), a vacuum was applied for 20 minutes to remove oxygen and other gases from the culture medium, and then a pressure of 30 psi was applied to the The container is filled with 5% carbon dioxide and 95% nitrogen for 1 minute. The oxygen content in the container was determined to be zero with an O ₂ indicator (FYRITE gas analyzer, Bacharach, Inc). Plates were left indoors for 6 hours. As an experimental control, duplicate culture plates were maintained under normal culture conditions (5% carbon dioxide and 95% ambient gas) for the same length of time. After 6 hours, the treated culture plate was removed from the container, and the stop solution containing 25mM glucose [supplemented with 1×sodium pyruvate, 10.0mM HEPES and 1×N2 supplemented DMEM] was used to replace the culture medium under hypoxic and normoxic conditions. medium, and cultured in the conditions of 5% carbon dioxide and 95% ambient gas. Neurons were treated with different concentrations of PAN-811 and vehicle as negative controls. MK801 was used as a positive control. Mitochondrial function and cell death were assessed by MTS and LDH assays (see below) 24 or 48 hours after H/H challenge.

In the hypoxia-only model, neurons were pretreated with vehicle or PAN-811 for 24 or 48 hours. Drug treatment continued during and after 24 hours of hypoxia. 24 or 48 hours after hypoxic challenge, cell morphology and function were evaluated (MTS and LDH analysis).

3.4.3. Morphological monitoring

In Figure 4, neuronal cell death is seen morphologically assessed. Before hypoxia, neurons were healthy with phase-brilliant soma (head of arrow) and intact neurites (open arrow). Background, these protrusions and their branches form a dense network. Hypoxia leads to shrinkage of cell bodies and collapse of neurites and networks. The 5μM dose of PAN-811 and the glutamate NMDA receptor antagonist MK801 effectively prevented the death of nerve cells and partially preserved neurites.

3.4.4.MTS analysis

The MTS assay is a colorimetric assay for the measurement of mitochondrial function in metabolically active cells. This detection indirectly reflects cell viability. The MTS tetrazolium compound is reduced in the mitochondria of metabolically active cells to a colored tissue culture medium-soluble formazan product whose absorbance at 490 nm can be detected. 20 μl of MTS reagent (Promega) was added to wells of a 96-well culture plate containing 100 μl of medium containing the samples. Incubate the plate under humidified, 5% carbon dioxide, 37°C for 1 to 2 hours until the color develops sufficiently. Absorbance at 490 nm was recorded with a Bio-Rad 96-well plate reader.

3.4.5. LDH analysis

The lactate dehydrogenase (LDH) assay is based on the reduction of NAD by the action of lactate dehydrogenase. The resulting reduced NAD (NADH) is used for the stoichiometric conversion of tetrazolium dyes. If aliquots of cell-free medium taken from differently treated cultures are analyzed, activity can be used as an indicator of relative cell death as well as cell membrane integrity. A 50 [mu]l aliquot of medium was transferred from each well of the 96-well plate tested to a well in an unused plate and supplemented with 25 [mu]l aliquots of mixed substrate, enzyme and dye (Sigma). After incubation at room temperature for 20 to 30 minutes, spectrophotometric measurements were performed at a wavelength of 490 nm.

3 results

4.1. Simple hypoxia model

3.1.1. Efficacy and Toxicity of PAN-811

Cortical neurons were pretreated with PAN-811 for 48 hours before hypoxia. PAN-811 remained during 24 hours of hypoxia and 48 hours after hypoxia. As shown in Figure 5, PAN-811 at a dose of 2 μM completely prevented cell death, but cytotoxicity occurred at 50 μM.

3.1.2. Comparison with other neuroprotective agents

The cortex was treated with 2 μM PAN-811, 1:80 green tea or 5 μM MK801 for 24 hours before, during and after hypoxia for 24 hours. Among the reagents tested, PAN-811 showed the highest potency, completely preventing neuronal cell death and mitochondrial dysfunction.

mild H/H model

3.1.3 PAN-811 protects neurons from mild H/H-induced neurotoxicity before and during challenge

Embryonic (E17) rat cortical neurons were cultured for 15 days and treated with PAN-811 and vehicle 24 hours before and during hypoxia/hypoglycemia (6 hours). Results were evaluated by MTS and LDH analysis 17 hours after receiving the challenge. At a concentration of 5 μM, PAN-811 completely protected hypoxia/hypoglycemia-induced mitochondrial dysfunction and neuronal cell death, while 1:1520 dilution of PEG:EtOH (equivalent to the vehicle in 5 μM PAN-811 amount), no such protection is formed.

Representative data are shown in Figure 6. Table 2 below summarizes the results of 6 experiments covering the concentration range 2-50 [mu]M.

Table 2

4.2.2. PAN-811 protects cells from mild H/H-induced neurotoxicity during, and especially after, insult

After 15 days of neuron culture, they were treated with PAN-811 or vehicle PEG:EtOH (7:3) for 24 hours before 6 hours of H/H (previous group). Alternatively, after 16 days of neuron culture, the above reagents were treated during 6 hours of H/H (period group), during 6 hours of H/H and 48 hours after H/H (period and after group), or during 48 hour phase treatment after H/H (after group). 48 hours after the H/H phase, LDH analysis was performed. The results in Figure 7 show that PAN-811 protected neurons from death during H/H, especially after H/H, but the protection was negligible when neurons were treated before H/H.

3.2 Extreme H/H model

PAN-811 had no neuroprotective effect at a concentration of ≤50 μM (data not shown).

4 Conclusion

4.1. At 2μM, PAN-811 has a complete protective effect on neurotoxicity induced by simple hypoxia and mild H/H. PAN-811 at 100 μM only partially prevents extreme H/H-induced neuronal cell death. Therefore, PAN-811 is unlikely to be involved in energy metabolism.

4.2. When administered during or after hypoxic or ischemic insults, PAN-811 significantly protected neuronal cells from death.

4.3. PAN-811 was significantly more potent than MK801 and/or green tea.

4.4. PAN-811 at 50 [mu]M was neurotoxic in case of prolonged exposure (120 hours).

5.0 References

1. Jiang, Z.-G., Piggee, C.A., Heyes, M.P., Murphy, C.M., Quearry, B., Zheng, J., Gendelman, H.E., and Markey, S.P. Glutamate is a principal mediator of HIV-1-infected Immune competent human macrophage neurotoxicity (Glutamate is the main mediator of neurotoxicity of immunocompetent human macrophages infected by HIV-1). J. Neuroimmunology 117(12): 97-107, 2001.

B.K.N-tert-butyl-phenylnitrone improves recovery of brain energy state in ratsfollowing transient focal ischemia(N-叔丁基苯基硝酮改善大鼠在短暂的局部缺血后脑能量状态的恢复).Proc.Natl.Acad.Sci.USA 92：5057-5061，1995.2. Folbergrová, J., Zhao, Q., Katsura, K., and

BKN-tert-butyl-phenylnitrone improves recovery of brain energy state in rats following transient focal ischemia (N-tert-butylphenylnitrone improves recovery of brain energy state in rats following transient ischemia).Proc.Natl.Acad. Sci. USA 92:5057-5061, 1995.

3. Touzani, O., Roussel, S., and MacKenzie, E.T. The ischemic penumbra. (Ischemic penumbra) Curr. Opin. Neurol. 14: 83-88, 2001.

Example 4

In an in vivo model of transient focal cerebral ischemia, PAN-811 exhibits significant neuroprotective effects

1. Introduction

Reducing neurological damage in the first few minutes after a stroke is an important strategy for achieving effective treatment. During a stroke, a thrombus emboli seals off an artery, interrupting oxygen and glucose supply to a local brain region, resulting in the loss of neurons in the infarct's central nucleus. Cells in the central nucleus die very rapidly by a necrotic mechanism. The brain area surrounding the ischemic infarction area, the structure is still there, but the (electrical conduction) function is terminated, which is called the penumbra. The penumbra is a transient area, and its evolution into infarction is a relatively progressive phenomenon (Touzani et al., 2001). This region offers the possibility to salvage some brain function, and the therapeutic window for the penumbra is substantially longer than for the infarct. The penumbra can also be described as an area of restricted blood supply, where energy metabolism remains. Thus, the penumbra is a target area for neuroprotective treatments and agents that reactivate quiescent neurons, such as hyperbaric oxygen. Thus, the immediate damage to the central nervous system may not be reversible, but aggravated brain damage, mainly the progression of a diffuse cerebral hypoxic/ischemic chain of events, can be prevented by effective neuroprotective strategies. For example, administration of neuroprotective agents before and during coronary artery bypass grafting (CABG) is effective in protecting against neurodegeneration caused by short-term changes in cerebral blood flow that cause a mild hypoxic/hypoglycemic state. Thus, compounds that can significantly protect nerves and rescue neurons after nerve damage are of great importance.

2. Purpose

In in vitro models of oxidative stress and ischemia, PAN-811 has shown significant neuroprotective effects. The existing work on this compound and the known toxicity profile and pharmacokinetic data strongly suggest the potential of PAN-811 for the treatment of stroke.

3.0 Materials and methods

3.1 Materials

-PAN-811 (Vion Pharmaceuticals)

- Ethanol (Sigma)

-PEG-300 (Sigma)

-MTS Assay Kit (Promega)

3.2 Abbreviations

CABG = coronary artery bypass surgery

EtOH = ethanol

H/H = hypoxia/hypoglycemia

MCAO = middle cerebral artery occlusion

PEG = polyethylene glycol

3.3 Research Design

3.3.1 In vitro studies PAN-811 was tested in several cellular models of neurodegeneration before initiating in vivo studies.

3.3.1.1. Neuron culture Enriched neuron cultures were prepared from 15-day-old Sprague-Dawley rat embryos. Using aseptic technique, mouse embryos are removed from the uterus and placed in sterile neuronal medium. Under a dissecting microscope, the brain tissue was removed from each embryo, and the meninges and blood vessels were carefully removed. The cerebellum was isolated by gross dissection under a microscope, and only cerebellar tissue was used for culture. Grind the tissue, dissociate the cells, and inoculate the poly-L-lysine precoated 48-well culture plate at a density of 5×10 ⁵ cells per well. Cultures were maintained in a solution containing equal amounts of Eagle's basal medium (without glutamine) and Ham's F-12k medium supplemented with 10% heat-inactivated horse serum, 10% fetal bovine serum, 600 μl /ml glucose, 100μg/ml glutamine, 50U/ml penicillin and 50μg/ml streptomycin. After 48 hours, 10 μM cytarabine was added to inhibit the division of non-neuronal cells. Cells cultured for 7 days were used for experiments.

3.3.1.2. Neurotoxicity of PAN-811 Cells were treated with different concentrations (0-100 μM) of PAN-811 for 24 hours. Cell viability was determined by MTT assay.

3.1.3.3. Induction of neurotoxicity in in vitro models Four in vitro models of excitotoxicity were studied. Cells were exposed to H/H conditions for 3 hours, or treated with 100 μM glutamate, 1 μM staurosporine, 10 μM veratridine for 45 minutes. All cells were co-treated with or without 10 [mu]M PAN-811 in Locke's solution. At the end of each excitotoxic exposure, the conditioned medium (original medium) was replaced. H/H was induced by incubating the cells in Locke's solution without added glucose in a humidified airtight container saturated with 5% carbon dioxide and 95% nitrogen for 3 hours.

3.1.3.4. MTS analysis After 24 hours of excitotoxicity, the cell viability was evaluated. A tetrazolium salt based on 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium salt (MTT, SigmaChemical Co. Ltd., St. Louis, Mo) was used Colorimetric assay for quantitative evaluation of cell damage. Briefly, dye was added to each well (final concentration 1.5 mg/ml), cells were incubated with MTT-acidified isopropanol (0.1 N HCl in isopropanol), and each sample was assayed in a 96-well plate reader for Absorption intensity at 540nm. Values are expressed as percentage of vehicle-treated control cells maintained on each plate, and percent change in cell viability was calculated.

3.1.2 In vivo studies

3.1.2.1. MCAO Thirty-six male Sprague-Dawley rats (270-330 g, Charles River Labs, Raleigh, VA) were used in this study. Anesthesia was induced with 5% halothane and maintained in 2% halothane in oxygen. Body temperature was maintained at normal temperature (37±1°C) throughout the operation with a constant temperature heating system (Harvard Apparatus, South Natick, MA). Food and water were ingested ad libitum before and after the operation, and the animals were reared in separate cages in a 12-hour light-dark cycle environment. After anesthesia, transient ischemia and reperfusion were achieved using the filament method of middle cerebral artery occlusion (MACO). Briefly, the right external carotid artery was isolated and its branches were coagulated. A 3-0 uncoated round-tipped monofilament nylon suture was inserted through the external carotid artery into the internal carotid artery and advanced (approximately 22 mm from the carotid bifurcation) until slight resistance was encountered, thus sealing the MCA the beginning. The intravascular sutures were left in place for 2 hours and then retracted to allow blood reperfusion to the MCA. After MCAO surgery, animals were placed in recovery cages maintained at a room temperature of 22°C. A 75 W heat lamp was also placed directly on top of each cage during the 2-hour ischemic phase and for the first 6 hours post-ischemic to ensure that the body temperature of the animals remained constant at normothermia throughout the experiment.

3.1.2.2. Treatment with PAN-811 Rats were treated with 1 mg of PAN-811 per kg body weight by intravenous injection for 10 minutes prior to MCAO. A stock solution of PAN-811 in 70% PEG300, 30% EtOH was prepared. This stock solution was diluted 5-fold with sterile saline (final concentration 1 mg/ml) prior to injection.

3.1.2.3. Determination of infarct volume Analysis of ischemic brain injury was measured as a function of total infarct volume for each rat brain. This was obtained from 7 coronal sections (2 mm thick) stained with 2,3,5-triphenyltetrazolium chloride (TTC). The brain slice collection area starts at 1 mm from the frontal pole and ends at the rostral end of the corticocerebellar junction. Infarct volumes were calculated using computer-assisted image analysis. Briefly, the posterior surface of each TTC-stained forebrain section was digitally imaged (Loats Associates, Westminster, MD) and the area of ischemic injury (in square millimeters) quantified.

4 results

4.1. In vitro studies

4.1.1 The neurotoxicity results of PAN-811 are shown in Figure 1. In fact, PAN-811 was only mildly toxic at concentrations up to 100 μM. At the highest concentration tested, the maximum toxicity was only 8.7% (see Figure 8).

4.1.2 Neuroprotection of PAN-811 PAN-811 was shown to significantly protect neurons from different excitotoxic agents ( FIG. 2 ). Neurons were pretreated with 10 μM PAN-811 to protect against hypoxia/low glucose (protection about 92%), 100 μM glutamate (protection about 75%), 1 μM staurosporine protein kinase C inhibitor and cell apoptosis inducer (protection about 47%) %) and 10 μM veratridine sodium channel blocker (about 39% protection) to treat the damage induced by 3 hours (see Figure 9).

4.2 In vivo studies

The results of this experiment are shown in Table 3. Thirty-six rats were used in this experiment, while 11 rats were excluded due to the following reasons: 4 died of severe stroke without bleeding complications, 4 developed subacute hemorrhage (SAH ) (3 died within 24 hours), 1 due to a sudden fire drill during the operation, 1 due to statistics as an irrelevant item, and 1 due to overdose of halothane. Of the 7 rats that died (4 without SAH, and 3 with SAH died of severe stroke), 6 were untreated (vehicle) rats and only 1 was PAN-811 treated. The mean infarct volume of vehicle-treated rats was 292.96 mm ³ , with a range of 198.75-355.81. The mean infarct volume of PAN-811 treated rats was 225.85 mm ³ , ranging from 42.36 to 387.08. This showed 23% neuroprotection (p<0.05). For unknown reasons, more severe damage than normal measurements was observed in the control group. Consequently, infarct size in PAN-811-treated animals was also much more neuroprotective than predicted. Nonetheless, the variability between the two groups was excellent (SEM of 10% or less), and the variability results were as good as, if not better than, previously published studies.

5 Conclusion

11.1 PAN-811 was well tolerated and relatively nontoxic in both in vitro and in vivo model systems.

11.2 Pretreatment of neurons with 10 μM PAN-811 significantly protected neurons from excitotoxicity leading to neurodegeneration.

11.3 Pretreatment of rats with a single dose (1 mg/ml) of PAN-811 10 minutes before transient ischemia can reduce the average infarct volume by 23%.

6 References

6.1 Literature

5.1.1 Williams AJ, Dave JR, Phillips JB, Lin Y, McCabe RT, and Tortella FC. (2000) Neuroprotective efficacy and therapeutic window of the high-affinity N-methyl-D-aspartate antagonist conantokin-G: invitro(primary neurosarbellar ) and in vivo (rat model of transient focalbrain ischemia) studies (Neuroprotective efficacy and therapeutic window of the high-affinity N-methyl-D-aspartate antagonist conantokin-G: in vitro (primary cerebellar neurons ) and in vivo (transient cerebral ischemia rat model) studies). J Pharmacol Exp Ther.294(1): 378-86.

7. Table 3

Table I: Infarct volumes in vehicle and PAN-811 treated rats. Rats were treated with 1 mg/ml PAN-811 for 10 minutes before MACO, and the infarct volume was measured 24 hours after surgery.

Example 5

PAN-811 protects neurons from hydrogen peroxide-induced oxidative stress

1.0 purpose

The aim of this study was to evaluate the efficacy of PAN-811 as a neuroprotective agent in a cellular model of oxidative stress associated with Alzheimer's disease. Neuroprotection and cytotoxicity were determined. Different solvents were tested to determine their suitability as delivery vehicles for PAN-811.

2.0 Materials and methods

2.1 Materials

- Neural Basal Medium (Invitrogen)

-B27-AO (Invitrogen)

-PAN-811 (Vion Pharmaceuticals, Inc.)

- Hydrogen peroxide (Calbiochem)

- Ethanol (Sigma)

-DMSO (Sigma)

-PEG-300 (Sigma)

-MTS Assay Kit (Promega)

2.2 Equipment

- Balance (Mettler-Toledo, Inc.)

-Adjustable sampler (Finnpipette)

-Cell culture cabinet (Thermo Forma)

-Cell incubator (Thermo Forma)

- Plate reader (Bio-Rad Model 550)

2.3 Research Design

2.3.1 Isolation and culture of cells

Primary cortical neurons were isolated from embryonic brains of 17-day-old rats, seeded into 96-well plates at 50,000 cells per well, and cultured in conventional neural basal medium for 2-3 weeks. Replace the medium twice with half volume of fresh neural basal medium without antioxidants.

2.3.2 Treatment with PAN-811 and hydrogen peroxide

PAN-811 was dissolved in ethanol or DMSO at 1 mg/ml (~5 mM), in PEG-300/ethanol (70%/30%) at 5 mg/ml (~25 mM), and further diluted to 1 μM with medium , 5 μM, 20 μM and 50 μM final concentrations. Neurons were treated with PAN-811 or vehicle for 24 hours prior to oxidative stress induced by hydrogen peroxide (60-70 [mu]M final concentration). Controls included untreated cells (no PAN-811 and hydrogen peroxide treatment), cells treated with PAN-811 alone, and cells exposed to hydrogen peroxide without PAN-811. Untreated cells were used as controls to assess neuronal toxicity and improved survival. Each analysis was repeated 3 times. Equal volumes of solvents (ethanol, DMSO and PEG-300/ethanol) were added to the cells to determine the effect of solvents on the assay.

2.3.3. Evaluation of cell function

After 24 hours, cultures were assessed for viability and mitochondrial function using a standard MTS assay (Promega). Follow manufacturer's operating instructions.

3. Results

3.1 Experiment 1

Experiments were performed as described in the study design above. At the end of treatments, all treatments and media were replaced with 100 [mu]l fresh pre-warmed Neurobasal media plus B27(-AO). The culture plate was returned to the cell incubator at 37°C, 5% CO ₂ for one hour, then 20 μl of MTS reagent was added to each well, and the culture plate was incubated in the cell incubator at 37°C, 5% CO ₂ for two hours. The absorbance of each well at 490 nm was recorded with a BioRad plate reader (model 550). Wells containing medium only served as blank controls. Each data point is the average of three individually analyzed wells. Untreated cells were used as a control to calculate cell viability and neuroprotection. Three week old primary cultures were used for this study. See Figure 10 for the results.

3.2 Experiment 2

The experiment was carried out in the same way as Experiment 1. Two-week-old primary cultures were used for this study. See Figure 11 for the results.

4. Discussion

All three solvents had minimal impact on the assay system at dilutions corresponding to a final PAN-811 concentration of 1-10 μM. DMSO showed a certain level of neuroprotection at dilutions corresponding to a final concentration of PAN-811 of 20 μM or greater. Ethanol and PEG-300/ethanol showed a certain level of neuroprotection at a dilution equivalent to a final concentration of 50 μM of PAN-811. PAN-811 showed good neuroprotection at 1-10 μM. Compared with ethanol alone, PAN-811 has better solubility in PEG-300/ethanol.

5 Conclusion

PAN-811 showed good neuroprotective ability at the final concentration of 1-10 μM. PEG-300/ethanol has the least interference on the analysis system when it is equivalent to the dilution of PAN-811 1-20μM, so it is the best solvent for PAN-811 among the three solvents tested.

It will be readily apparent to those skilled in the art that the present invention is well adapted to carry out the objects and attain the end result and advantages stated and inherent therein. It will be apparent to those skilled in the art that various modifications and changes can be made in practicing the present invention without departing from the spirit and scope of the present invention. Modifications and other applications described herein may occur to those skilled in the art within the scope encompassing the spirit of the invention as defined in the claims.

Claims (1)

1. The application of the compound of the following formula in the preparation of medicines for the treatment of ischemia-related diseases: Wherein the ischemia-related disease is selected from the group consisting of: Alzheimer's disease, Parkinson's syndrome, neurodegeneration caused by short-term changes in blood flow in the brain during coronary artery bypass surgery, diffuse cerebral ischemia caused by cardiac arrest Blood, focal cerebral infarction, cerebral hemorrhage, hemorrhagic infarction, hypertensive hemorrhage, hemorrhage caused by abnormal rupture of intracranial blood vessels, subarachnoid hemorrhage caused by rupture of intracranial aneurysm, hypertensive encephalopathy, carotid artery stenosis or obstruction, cardiogenic thromboembolism, spinal cord stroke, spinal cord injury, atherosclerosis, vasculitis, plaque degeneration, myocardial infarction, myocardial ischemia, and supraventricular tachycardia.

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