KR20100100858A - Methods of identifying safe nmda receptor antagonists - Google Patents

Abstract

The present invention provides a method of treating a disease that lowers pH in an area of infected tissue, comprising analyzing a potency boost, or potency boost, of a compound at disease-induced and physiological pH in cells expressing human NMDA receptors. Or to a method of identifying a compound useful for prophylaxis. Assessment of potency boost was performed at least 5 times until the compound's IC ₅₀ (“potency boost”) at physiological pH and disease induced pH, until the 95% confidence interval for potency boost did not change more than 15% with the addition of a new experiment. It includes measuring repeatedly. The method may be useful in the selection of safe NMDA receptor antagonists for the treatment or prevention of diseases that lower pH in the area of infected tissue. Such diseases include, but are not limited to, neuropathic pain, ischemia, Parkinson's disease, epilepsy and traumatic brain injury.

Description

METHODS OF IDENTIFYING SAFE NMDA RECEPTOR ANTAGONISTS

Cross Reference of Related Applications

This application discloses US Provisional Application 60 / 985,922, filed November 6, 2007, entitled “Methods of Identifying Safe NMDAR Antagonists,” and US Provisional Application 60 / 985,924, filed November 6, 2007. The name, "Methods of Identifying Safe NMDAR Antagonists to Treat Neuropathic Pain," is hereby prioritized, and is hereby incorporated by reference in its entirety.

Field of invention

The present invention relates to an improved method of selecting a safe and effective pH dependent N-methyl D-aspartate receptor antagonist used as a means to prevent or minimize tissue damage before, during and after a pH-lowering phenomenon. .

Neurons, or neurons, transmit signals from the periphery to the central nervous system (CNS), between different regions of the CNS, and from the CNS to other organs (ie, peripherals). This signal transduction is basically mediated by small molecules called neurotransmitters. In general, neurotransmitters can be classified as irritant or inhibitory. Stimulating neurotransmitters increase the activity (eg, firing rate) of signal receiving (ie, postsynaptic) neurons and decrease inhibitory neurotransmitters. Neurons differ in their ability to recognize, integrate, and pass signals transmitted by neurotransmitters. For example, some neurons may ignite at a constant rate, stimulating or suppressing changes in surroundings. Other neurons are typically at rest in the absence of external stimulation. Thus, any modification of these activities occurs in the form of stimuli. After all, neuronal stimulation plays a fundamental role in controlling brain function. Of the large molecules that govern general brain function, glutamate (also called glutamic acid) is one of the most important. Research into its function has made significant progress in understanding how the brain works. The role of glutamate as an important signal molecule has only been revealed in the last 20 years.

Glutamate is an amino acid. As other amino acids, glutamate is present throughout the brain in relatively high concentrations. As a result, the researchers initially thought that glutamate was a mediator metabolite of several cellular responses that were not originally associated with neuronal signal transduction, and did not attempt to interpret their presence in neurons as evidence of their potential role as neurotransmitters. It was. In the 1950s, the first signs of glutamate's stimulatory function in the brain were highlighted, but initially, glutamate applied to neurons elicited stimulatory responses in virtually all areas of the brain examined; As such, these findings were ruled out. Only afterwards, the scientists thought that they could be due to the activity of the stimulatory receptors present throughout the CNS, and found that the effect of the observed glutamate was really justified. In the 1970s and 1980s, researchers recognized that certain glutamate receptors, ie proteins on the surface of neurons that specifically bind glutamate, are secreted by other neurons, thereby inducing stimulation of posterior synaptic neurons. Was done. Recognition of these glutamate receptors has emphasized the importance of glutamate as a stimulating neurotransmitter.

Knowledge of glutamatergic synapses has developed significantly over the past decade, among other things, through the application of molecular biological techniques to the study of glutamate receptors and transporters. Presently, it has an intrinsic cation permeable channel, N-methyl-D-aspartate, alpha-amino-3-hydroxy-5methyl-4-isoxazolepropionic acid (AMPA) and catenate receptor It is known that there are three family of ion channel receptors. In addition, three groups of metabolic, G protein-conjugated proteins that modify neurons and glia stimulation through membrane ion channels and G protein subunits that act on secondary transporters such as diacylglycerol and cAMP ) Glutamate receptor (mGluR). In addition, there are two glia glutamate transporters and three neuronal transporters in the brain.

Glutamate is essential for normal brain function. Glutamate plays a primary role in controlling cognition, motor function, synaptic plasticity, learning and memory. High levels of endogenous glutamate can cause brain damage due to overactivation of NMDA, AMPA or mGluR1 receptors. Examples of brain damage associated with excess glutamate or irritant toxicity appear after overlapping epilepsy, cerebral ischemia, and traumatic brain injury. Stimulating toxicity (eg, toxicity from overactivation of glutamate receptors) can also cause chronic neurodegeneration, such as Parkinson's disease, Alzheimer's disease, Lou Gehrig's disease and Huntington's chorea. In animal models of cerebral ischemia and traumatic brain injury, NMDA and AMPA receptor antagonists protect against severe brain injury and delay behavioral deficits. Other clinical conditions that may respond to drugs acting on glutamate delivery include epilepsy, amnesia, anxiety, hyperalgesia and psychosis (Meldrum BS. J Nutr. 2000 Apr; 130 (4S Suppl): 1007S-15S).

NMDA Receptor Antagonist

NMDA subtypes of glutamate-gated ion channels mediate stimulatory synaptic transmission between neurons in the central nervous system (Dingledine et al. (1999), Pharmacological Reviews 51: 7-61). NMDA receptors consist of NR1, NR2 (A, B, C and D), and NR3 (A and B) subunits, which determine the functional properties of innate NMDA receptors. Expression of the NR1 subunit alone does not result in functional receptors; Co-expression of one or more NR2 subunits is required to form a functional channel. In addition to glutamate, NMDA receptors require a co-agonist, glycine, to bind to make the receptor work. Glycine binding sites are found in the NR1 subunit, while glutamate binding sites are found in the NR2 subunit. The NR3 subunit also binds glycine. The NR2B subunit also has a binding site for spermine-like polyamine, a regulatory molecule that regulates the function of the NMDA receptor. At resting membrane potential, the NMDA receptor is very inactive. This is due to the voltage dependent barrier of the channel pores by magnesium ions which prevents ion flow through the pores. Depolarization removes the channel barrier and allows activated NMDA receptors to flow ionic current through the posterior synaptic membrane. NMDA receptors can transmit not only calcium ions but also other ions. NMDA receptors are regulated by many endogenous and exogenous compounds. Likewise, sodium, potassium and calcium ions not only pass through the NMDA receptor channel but also regulate the activity of the NMDA receptor. Zinc blocks NMDA currents through NR2A-containing receptors in a noncompetitive, high affinity and voltage dependent manner. It has also been demonstrated that polyamines act to enhance or inhibit glutamate-mediated responses, rather than directly activating the NMDA receptor.

In animal models of stroke and brain injury, glutamate released from infected neurons has been found to overstimulate NMDA receptors, which can lead to neuronal death. Thus, compounds that block NMDA receptors have been considered to be therapeutics for stroke or brain injury. In recent animal studies, NMDA receptors have been identified as targets for neuroprotection in related settings implicated in stroke, brain and spinal cord injury, and cerebral ischemia. NMDA receptor blockers are effective against limited volumes of damaged brain tissue in experimental models of stroke and traumatic brain injury (Choi, D. (1998), Mount Sinai J Med 65: 133-138; Dirnagle et al. (1999) Tr. Neurosci. 22: 391-397; Obrenovitch, TP and Urenjak, J. (1997) J Neurotrauma 14: 677).

Numerous NMDA receptor antagonists have been tested in early clinical trials for stroke. Stroke is the third leading cause of death in the United States and the most common cause of adult disease. Ischemic stroke occurs when the blood vessels in the brain are blocked, which blocks the flow of blood to each part of the brain. Tissue plasminogen activator (TPA), currently the only approved stroke treatment, is a thrombolytic that promotes the dissolution of blood clots in blood vessels. Neuroprotective agents were as much as interested in treating thrombolytics (http://www.emedicine.com/neuro/topic488.htm, Lutsep & Clark "Neuroprotective Agents in Stroke", April 30, 2004). However, no human treatment has been approved.

The most commonly studied neuroprotective agents of acute stroke block the N-methyl-D-aspartate (NMDA) receptor. Structural analogs of dextrorphan, NMDA channel blockers and antitussives are one of the first NMDA antagonists studied in human stroke patients. Unfortunately, dextropan causes hypotension as well as hallucinations and excitability, limiting its use (Albers et al. Stroke (1995) 26: 254-258). Selfotel, a competitive NMDA antagonist, tended to show higher mortality in treated patients than the placebo-treated cohort, so the trial was discontinued quickly. Testing of other NMDA receptor antagonists, Aptiganel HCl (Cerestat), was discontinued due to concerns about the risk ratio for gains. In an attempt to avoid this adverse effect, indirect NMDA receptor antagonists have been developed that act on the glycine site of the receptor. These agents prevent glycine from binding and inhibit glutamate from activating the receptor. Early clinical trials have shown that less glycemic side effects occur with these glycine site NMDA antagonists. In a huge 1367 patient group, efficacy test of drug GV150526 was completed in 2000. It was reported that the drug was safe and well tolerated, but no improvement was observed in any of the three month performance measures (http://www.emedicine.com/neuro/topic488.htm, Lutsep & Clark "Neuroprotective Agents in Stroke ", April 30, 2004).

Epilepsy has been considered as a potential therapeutic target for glutamate receptor antagonists. Of course, at therapeutic concentrations, the common anticonvulsant valproate may act in part as an anticonvulsant by blocking the AMPA receptor. NMDA receptor antagonists are known as anticonvulsants in many experimental models of epilepsy (Bradford (1995) Progress in Neurobiology 47: 477-511; McNamara, JO (2001) Drugs effective in the therapy of the epilepsies.In Goodman & Gliman's: The pharmaco logical basis of therapeutics [Eds. JG Hardman and LE Limbird] McGraw Hill, New York).

NMDA receptor antagonists may be useful for the treatment of chronic pain. Chronic pain caused by damage to peripheral or central nerves is often proven to be very difficult to treat, even with opioids. Treatment of chronic pain with ketamine and amantadine has proven useful, and the analgesic effects of ketamine and amantadine are known to be mediated by the blocking of NMDA receptors. In some reports, systemic administration of amantadine or ketamine has been shown to substantially reduce the intensity of trauma caused by neuropathic pain. In small-scale, double blind, randomized clinical trials, amantadine significantly reduced neuropathic pain in cancer patients (Pud et al. (1998), Pain 75: 349-354), and ketamine caused peripheral nerves. Injury (Felsby et al. (1996), Pain 64: 283-291), patient group with peripheral vascular disease (Perrson et al. (1998), Acta Anaesthesiol Scand 42: 750-758), or kidney donor It has been demonstrated to reduce pain in the patient group (Stubhaug et al. (1997), Acta Anaesthesiol Scand 41: 1124-1132). "Wind-up pain" caused by repeated pinpricking has also been significantly reduced. These findings indicate that central sensitization caused by nociceptive input can be prevented by administration of NMDA receptor antagonists.

NMDA receptor antagonists are also useful for the treatment of Parkinson's disease (Blandini and Greenamyre (1998), Fundam Clin Pharmacol 12: 4-12). Amantadine, an anti-Parkinson's disease drug, is an NMDA receptor channel blocker (Blanpied et al. (1997), J Neurophys 77: 309-323). Amantadine is a serum used alone because of limited efficacy. However, small scale clinical trials have demonstrated the value of amantadine as an additional treatment with L-DOPA. Amantadine reduced the severity of dyskinesia by 60% in these patient groups without reducing the anti-Parkinson's effect of L-DOPA itself (Verhagen Metman et al. (1998), Neurology 50: 1323-1326). Similarly, another NMDA receptor antagonist, CP-101,606, has been shown to reduce the possibility of Parkinson's syndrome caused by L-DOPA in monkey models (Steece-Collier et al., (2000) Exper. Neurol, 163: 239-243).

NMDA receptor antagonists are also useful for the treatment of brain cancer. Fast-growing brain glioma is a cellular component that kills adjacent neurons by secretion of glutamate and overactive NMDA receptors, just as dying neurons provide room for growing tumors. Can emit. Many studies have shown that NMDA receptor antagonists can reduce tumor growth rates in vivo as well as in some in vitro models (Takano, T., et al. (2001), Nature Medicine 7: 1010-1015; Rothstein, JD and Bren, H. (2001) Nature Medicine 7: 994-995; Rzeski, W., et al. (2001), Proc. Nat'l Acad. Sci 98: 6372).

While NMDA receptor antagonists are useful in the treatment of many intractable diseases, to date, dose limiting side effects have prevented the clinical use of NMDA receptor antagonists for these conditions. The first three occurrences of NMDA receptor antagonists (competitive blockers at channel blockers, glutamate or glycine antagonist sites, and noncompetitive allosteric antagonists) are clinically useful due to toxic side effects, such as psychotic symptoms and cardiovascular effects. Not proven. In particular, cardiovascular side effects (hypotension and hypertension) were most prominent and dose limiting in many small scale human studies. In addition, administration of NMDA antagonists resulted in undesirable effects on memory and attention. In addition, NMDA receptor antagonists such as ketamine cause psychotic conditions reminiscent of schizophrenic symptoms in humans (Krystal et al. (1994), Arch Gen Psychiatry 51: 199-214). In addition, ataxia, cognitive impairment, dyskinesia, excitement, confusion, dizziness and hypothermia all result from the administration of NMDA antagonists. Thus, despite the excellent potential of glutamate antagonists in the treatment of many serious diseases, the severity of the side effects has given up hope for the development of resistant NMDA receptor antagonists (Hoyte L. et al (2004) "The Rise and Fall of NMDA Antagonists for Ischemic Stroke Current Molecular Medicine "4 (2): 131- 136; Muir, KW and Lees, KR (1995) Stroke 26: 503-513; Herrling, PL, ed. (1997)" Excitatory amino acid clinical results with antagonists "Academic Press; Parsons et al. (1998) Drug News Perspective II: 523 569).

pH Sensing NMDA Receptor

In the late 1980s, new properties of NMDA receptors were discovered, and more recently, new classes of NMDA antagonists have been developed. The two most common subtypes of NMDA receptors typically have special properties of about 50% inhibition by both at physiological pH (Traynelis, S.F. and Cull-Candy, S.G. (1990) Nature 345: 347). Inhibition of NMDA receptors by both is controlled by NR2B subunits and selective exons splitting in NR2A and NR1 subunits (Traynelis et al. (1998), J Neurosci 18: 6163-6175). .

Extracellular pH is very dynamic in the mammalian brain and affects the action of many biochemical processes and proteins, including glutamate receptor action. PH-sensing of the NMDA receptor is of interest for at least two reasons. First, the IC ₅₀ value for proton inhibition of pH 7.4 places the receptor under sustained inhibition at physiological pH. Second, pH changes are widely documented in the CNS during synaptic transmission, glutamate receptor activity, glutamate receptor uptake, ischemia and seizures (Siesjo, BK (1985), Progr Brain Res 63: 121-154; Chesler, M (1990) Prog Neurobiol 34: 401-427; Chesler and Kaila (1992), Trends Neurosci 15: 396-402; Amato et al. (1994), J Neurophysiol 72: 1686-1696). Acidification associated with this latter pathological condition can inhibit NMDA receptors, which provide negative feedback that minimizes their contribution to neurotoxicity (Kaku et al. (1993), Science 260: 1516-1518; Munir and McGonigle (1995), J Neurosci 15: 7847-7860; Vornov et al. (1996), J Neurochem 67: 2379-2389; Gray et al. (1997), J Neurosurg Anesthesiol 9: 180- 187; but see O'Donnell and Bickler (1994), Stroke 25: 171-177; reviewed by Tombaugh and Sapolsky (1993), J Neurochem 61: 793-803) and seizure maintenance (Balestrino and Somjen (1988), J Physiol (Lond) 396: 247-266; Velisek et al. (1994), Exp Brain Res 101: 44-52). This feedback inhibition can also delay the contribution of NMDA receptor activity to ischemic cell death until the pH gradient is restored before glutamate is removed from the interstitial space. PH sensing of glutamate uptake may increase the chance of post-insult treatment such as stroke with NMDA receptor antagonists (Tombaugh and Sapolsky (1993), J Neurochem 61: 793-803). (Billups and Attwell (1996), Nature (Lond) 379: 171-173).

Some diseases cause a sharp decrease in pH. For example, in stroke, transient ischemia reduces the pH to 6.4-6.5 in the infarct nuclear region, with a slight decrease in the region around the nucleus. The penumbral region, which surrounds the nucleus and extends outward, suffers significant neuronal loss. The pH in this region drops to about pH 6.9. Reduced pH induction is noticeable in the presence of excess glutamate and is attenuated in hypoglycemic conditions (Mutch & Hansen (1984) J Cereb Blood Flow Metab 4: 17-27; Smith et al. (1986) J Cereb Blood Flow Metab 6: 574). -583; Nedergaard et al. (1991) Am J Physiol 260 (Pt3): R581-588; Katsura et al (1992a) Euro J Neursci 4: 166-176; and Katsura & Siesjo (1998) "Acid base metabolism in ischemia in pH and Brain function (Eds Kaila & Ransom) Wiley-Liss, New York et al.).

In addition to ischemia, there are additional examples of various conditions in which the pH changes under general and special conditions that can be easily treated with NMDA antagonists. Typically, tissue extracellular pH is generally acidic than cerebrospinal fluid because of the regulation of both, as well as the active and passive movement of metabolites. It is known that various acid and alkali changes of epidemiologically active dependence in extracellular pH occur for almost 20 years. These changes are explained over a wide range of preparation and brain regions. This involves multiple molecular mechanisms, which include metabolic changes, lactic acid secretions, bicarbonate divergence through anion channels, Na ⁺ / H ⁺ and Ca ²⁺ / H ⁺ exchanges, and both releasing from acidified vesicles. Include. These are dependent on extracellular buffer systems in the brain of mammals that are heavily dependent on bicarbonate. Thus, the magnitude of the observed pH change is often dependent on the ability of the CNS tissue to rapidly interconvert bicarbonate-CO ₂ . Enzymes that do this (carbonic unhydrases), in turn, are instrumental in setting the level of pH change that can be achieved.

Neuropathic pain is associated with pH changes in the spinal cord. For example, applying a single electrical stimulus to spinal cord isolated from rat pups results in an alkaline shift of 0.05 pH units and a 0.1 pH shift after 10 Hz stimulation. When stimulation is stopped, acidification occurs, and this acidification is greater in older animals (Jendelova & Sykova (1991) Glia 4: 56-63). In addition, 30-40 Hz stimulation at the frog's dorsal root results in transient extracellular acidification in vivo, leading to a decrease in the maximum upper limit of 0.25 pH units at the lower dorsal horn. Extracellular pH changes increase with stimulus intensity and frequency (Chvatal et al. (1988) Physiol Bohemoslov 37: 203-212). In addition, high frequency (10-100 Hz) nerve stimulation in adult rat spinal cord in vivo causes three-phase alkali-acid-alkali shifts at extracellular pH (Sykova et al. (1992) Can J Physiol Pharmacol 70: Suppl S301- 309). It has also been shown that acute invasive stimuli (pinch, pressure, heat) applied to rat hindpaws cause transient acidification of 0.01-0.05 pH units at lower in vivo olfactory (lamina III-VII). Chemical or thermal damage caused a long term two-hour decrease in intercellular pH in the 0.05-0.1 pH unit. High frequency nerve stimulation caused an alkaline pH shift followed by a significant 0.2 pH unit acid shift (Sykova & Svoboda (1990) Brain Res 512: 181-189). Thus, increased firing of pain fibers can reduce (acidify) the pH of the spinal cord's sense of smell. Such acidification can increase the efficacy of pH dependent blockers in areas that make them useful in the treatment of acute nerve injury or acute pain syndrome.

Hypothalamic neurons are overactive in Parkinson's disease, which leads to lower pH in the local area. This decrease in pH increases the efficacy of the pH-sensitive antagonist in this region. In addition to the activity causing acidification, neuronal activity and extracellular pH correlate in the brain region. High frequency stimulation to brain slices is initially acidified, followed by alkalizing, then slowly acidifying (Chesler (1990) Prog Neurobiol 34: 401-427, Chesler & Kaila (1992) Tr Neurosci 15: 396-402, and Kaila & Chesler (1998) "Activity evoked changes in extracellular pH" in pH and Brain function (eds Kaila and Ransom). See Wiley-Liss, New York et al.).

Acidification also occurs during seizures. Since NMDA antagonists are anticonvulsants, epilepsy is inert outside the spatial, but pH-sensing NMDA antagonists become targets that can effectively act as anticonvulsants, and seizures are temporarily calmed. In a wide range of formulations, electronic seizures alter extracellular pH. For example, a drop below pH 0.2-0.36 may occur in a cat fascia dentata or rat hippocampal CA1 or dentate during an electrical or chemically induced seizure. Larger degradations close to 0.5 can occur under hypoxic conditions. This finding is well accepted and has been repeated in many formulations (Siesjo et al (1985) J Cereb Blood Flow Metab 5: 47-57; Balestrino & Somjen (1988) J Physiol 396: 247-266; and Xiong & Stringer) (2000) J Neurophysiol 83: 3519-3524).

In addition, other forms of brain damage can cause acidification. "Spreading depression" is a term used to describe the slow movement of an electrically inactive wave that in turn causes many traumatic injuries to brain tissue. Proliferation inhibition may occur during (brain) concussion or migraine headaches. Acidic pH changes occur with suppression of pronouncedness. Systemic alkalosis can occur together through a decrease in the amount of total carbon dioxide (hypercarbonate), such as hyperventilation. Systemic esciosis may occur with dyspnea, increased gas exchange, or increased levels of carbon dioxide in the blood (hypercarbonate) under conditions that impair lung function. Diabetic ketoacidosis and laccidosis represent three of the most serious acute diabetic complications, which can cause brain acidification. In addition, fetal asphyxia occurs during birth at a rate of 25 per 1000 live births. It is associated with hypoxia and similar, but not identical, brain damage to ischemia.

Until 1995, it was not known whether the quantum-sensing properties of NMDA receptors could develop as targets for small molecule regulation of receptors that could be developed therapeutically. In Traynelis et al. (1995 Science 268: 873), it was first reported that small molecule spermine can release proton inhibition, thereby regulating the function of NMDA receptors. Spermine, a polyamine, shifts the pKa of a quantum sensor to an acidic value, reducing the degree of tonic inhibition, which indicates the potential for action at physiological pH (Traynelis et al. (1995), Science 268: 873 Kumamoto, E (1996), Magnes Res 9 (4): 317-327).

In 1998, a mechanism for the activity of phenylethanolamine NMDA antagonists associated with quantum sensors was discovered. Ifenprodil and CP-101,606 increase the sensitivity of the receptor to both, increasing the inhibitory of both. By shifting the pKa value for proton blocking of NMDA receptors to more alkaline values, ifenprodil binding causes more of the receptor to be quantized at physiological pH and eventually inhibited. In addition, tests of an in vitro model of NMDA-induced stimulatory toxicity in the initial culture of the rat cerebral cortex revealed that fenprodil is more potent at lower pH (6.5) than at higher pH (7.5). It became. The inventors speculate that there may be a context-dependent blocker that is used in the treatment of stroke and which is inactive at physiological pH but may be active at lower pH values occurring during ischemia (Mott et al. 1998 Nature Neuroscience 1 : 659).

Ifenprodil is neuroprotective in animal models of focal cerebral ischemia (Gotti et al. (1988), J Pharmacol Exp Ther 247: 1211-1221; Dogan et al. (1997), J Neurosurg 87 (6): 921-926). Ifenprodil showed neuroprotective properties in mammals after middle cerebral artery occlusion. Dogan et al reported a 22% reduction in infarct volume in rats, while Gotti et al. Reported a 42% reduction in infarct volume at higher doses in Kat's experiment. In addition, Gotti et al. Reported that the fenprodil derivative SL 82.0715 reduced infarct volume by 36-48% at higher doses in cat and rat experiments. Unfortunately, fippleprodil and several analogs thereof, such as eliprodil and haloperidol (Lynch and Gallagher (1996), J Pharmacol Exp Ther 279: 154-161; Brimecombe et al. (1998) , J Pharmacol Exp Ther 286 (2): 627-634, block certain serotonin receptors and calcium channels in addition to NMDA receptors, which limit their clinical utility (Fletcher et al. (1995), Br J Pharmacol 116). (7): 2791-2800; McCool and Lovinger (1995), Neuropharmacology 34: 621-629; Barann et al. (1998), Naunyn Schmiedebergs Arch Pharmacol 358: 145-152). In addition, ifenprodil analogs elliprodil increase cardiac repolarisation with inhibition of IKr (Lengyel et al. (2004) Br J Pharmacol. Aug 9 [Epub ahead of print]), ifenprodil and certain Analogs also inhibit calcium channels (Biton et al. (1994), Eur J Pharmacol 257: 297-301; Biton et al. (1995), Eur J Pharmacol 294: 91-100; Bath et al (1996), Eur J Pharmacol 299: 103-112). CP 101,606 (Menniti et al. (1997), Eur J Pharmacol 331: 117-126), Ro 25-6981 (Fischer et al. (1997), J Pharmacol Exp Ther 283: 1285-1292) and Ro 8-4304 ( Several more selective ifenprodil derivatives are being considered for clinical development, including Kew et al. (1998), Br J Pharmacol 123: 463-472).

In addition to these allosteric modulators, other NMDA antagonists have been shown to produce neuroprotective effects in animal models of local ischemia (Gill et al (1994) Cerebrovascular and Brain Metabolism Reviews 6: 225-256). These NMDA antagonists are divided into three functional classes: competitive blockers of glutamate binding sites, competitive blockers of glycine binding sites and channel blockers, which cause toxic side effects in humans or exhibit limited efficacy.

(i) Competitive NMDA antagonists at the glutamate site, such as Cellpotel, D-CPPene (SDZ EAA 494) and AR-Rl 5896AR (ARL 15896AR), have toxic side effects such as excitement, hallucinations, confusion and infertility (Davis et. (2000), Stroke 31 (2): 347-354; Diener et al. (2002), J Neurol 249 (5): 561-568); Such as paranoia and delusions (Grotta et al. (1995), J Intern Med 237: 89-94); Psychotic similar syndrome (Loscher et al. (1998), Neurosci Lett 240 (l): 33-36); Low treatment rate (Dawson et al. (2001), Brain Res 892 (2): 344-350); Amphetamine-like stereotype behaviors (Potschka et al. (1999), Eur J Pharmacol 374 (2): 175-187).

(ii) Glycine site antagonists, such as HA-966, L-701,324, d-cycloserine, CGP-40116, and ACEA 1021 cause toxic side effects including significant memory impairment and impaired motor function (Wlaz, P (1998), Brain Res Bull 46 (6): 535-540).

(iii) NMDA receptor channel blockers, such as MK-801 and ketamine, have toxic side effects, such as psychosis-like (Hoffman, DC (1992), J Neural Transm Gen Sect 89: 1-10); Cognitive impairment (free play, cognitive memory and attention loss; Malhotra et al (1996), Neuropsychopharmacology 14: 301-307); Schizophrenia-like syndrome (Krystal et al (1994), Arch Gen Psychiatry 51: 199-214; Lahti et al. (2001), Neuropsychopharmacology 25: 455-467).

WO 02/072542 of Emory University describes a class of pH-dependent NMDA receptor antagonists that exhibit pH sensitivity tested in in vitro and epilepsy experimental models using oocyte assays. However, in vitro data using Xenopus oocytes are subject to various parameters in IC ₅₀ measured for optimally selected compounds or lead compounds. In addition, the assay is limited to cell-based screens, making it difficult to analyze whether the pH is sufficiently largely lowered in infected ischemic tissues in vivo to observe the substantial effects of pH-dependent antagonists. In addition, ischemia is a tissue-based disease in vivo, especially with core and peripheral damage, so that the outer core of the pH dependent NMDA antagonist is effective to some extent, given that the drop in pH is radially reduced from the infarct core. I do not know. Eventually, NMDA receptor antagonists are known to induce psychosis and other consciousness-altering side effects, with increased neuroprotective activity caused by ischemic pH lowering observed the alleviating effect of pH-sensitive NMDA receptor antagonists and It is not known whether it is sufficient to avoid receptor-related side effects.

WO 06/023957 of Emory University discloses: (i) a 95% increase in efficacy of compounds at disease-induced low and physiological pH in cells expressing the NR1 / NR2A NMDA receptor and / or the NR1 / NR2B NMDA receptor. Iteratively evaluating the potency boost experiment until the confidence interval has not changed more than 10% by the addition of a new experiment; (ii) testing the compound in an animal model of transient local ischemia and repeating the experiment until the 95% confidence interval does not change by more than 10% with the addition of a new experiment to determine the effect of the compound on infarct volume; (iii) selecting a compound having at least 5 potency elevations in step (i) and selecting at least one compound having an infarct volume reduction of at least 30% in step (ii). It is.

In sum, in order to select an appropriate NMDA receptor antagonist that can be resistant in humans, the drug must effectively block the glutamate system during pathological conditions, avoiding toxic side effects and not sufficiently affecting the general function of glutamate neurotransmission. PH-dependent selective NMDA antagonists, which show greater affinity for NMDA receptors at lower pH in vitro, have also been challenged to predict whether they can provide sufficient response in vivo and serve as commercial drugs. While pH-dependent NMDA receptor antagonists have evolved, the appropriate properties of these drugs to accurately establish successful variables for selection as drugs for human clinical use have not yet been determined.

Detailed description for carrying out the invention

The present invention aims to provide an improved method of selecting a pH dependent N-methyl D-aspartate receptor antagonist which is used to prevent or minimize tissue damage before, during and after pH-lowering phenomena.

In particular, it is an object of the present invention to provide a method of identifying active compounds useful for preventing or treating neuropathic pain or ischemic injury.

It is also an aspect of the present invention to provide compounds, compositions and methods useful for preventing or treating pathogenic pH-lowering symptoms.

Summary of the Invention

For better human clinical performance, an improved identification method of improved NMDA receptor antagonists is described for treating or preventing pH-lowering diseases in infected tissue areas. Specifically, the method uses cells expressing human NMDA receptors to assess the potency difference of compounds that inhibit NMDA receptor activity at physiological pH against elevated pH potency of the compound, ie ex vivo, pathogenic pH. This method identifies compounds with improved safety and efficiency profiles throughout previously known techniques.

The inventors surprisingly found that the potency boost assessed in cells expressing human NMDA receptors, when compared to the use of cells expressing NMDA receptors from other mammals, selected an effective and safe NMDA receptor antagonist. It has been found to provide an improved method. In particular, the increase in pH potency obtained from non-human NMDA receptors, such as the rat NMDA receptor, is not predictable of the rise in pH potency obtained from human NMDA receptors. The safety of NMDA receptor antagonists arises from the lack of efficacy of the compounds at physiological pH. Thus, ideal compounds are those that have very low potency at physiological pH, but are very effective at pathological conditions with lower pH.

The methods provided herein can be used for the selection of safe NMDA receptor antagonists for treating or preventing diseases in humans that lower pH in the area of infected tissue. Such diseases include, but are not limited to, neuropathic pain, ischemia, Parkinson's disease, epilepsy and traumatic brain injury.

In one embodiment, there is provided a method of identifying a compound useful for treating or preventing a disease that lowers pH in an area of infected tissue, and in cells expressing human NMDA receptors, the difference in potency of the compound at disease induced and physiological pH (for example, IC ₅₀ of the IC ₅₀ in the low pH / disease induction at physiological pH) includes a method of analyzing. The analysis of potency boost is a method of measuring IC ₅₀ at physiological and disease induced pH, repeating at least 5 measurements until the 95% confidence interval for potency boost has not changed more than 15% with the addition of a new experiment (so-called , "Potency increase").

In certain embodiments, the method also includes identifying a compound having a potency boost in at least five such cells. In certain embodiments, the potency boost of a compound in such cells is at least 2, or at least 3, or at least 4, or more than when measuring the potency boost of the same compound in a cell expressing a non-human NMDA receptor At least 5, or at least 6, or at least 7, or at least 8, or at least 9, or at least 10 is high. In certain embodiments, the potency boost of a compound is at most 100 or at most 50 higher than when measuring the potency boost of the same compound in cells expressing NMDA receptors other than humans. In certain embodiments, the non-human NMDA receptor is a rat NMDA receptor.

In one non-limiting embodiment, the infected tissue is selected from brain tissue, tissue damaged with ischemia, pain, particularly tissue infected with neuropathic pain, and tissue infected with traumatic brain injury.

In some embodiments, the 95% confidence interval does not change by at least 10%, at least 8%, at least 5%, at least 4%, at least 3%, or at least 2% with the addition of a new experiment.

In another incidental embodiment, the potency boost experiment is repeated five times, at least six times, at least seven times, at least eight times, at least nine times, or at least ten times.

In another aspect of the invention, identification of compounds useful for treating or preventing pain disorders in an infected tissue region includes: (i) the inhibition of a compound that inhibits human NMDA receptors at disease-induced and physiological pH in cells expressing human NMDA receptors. Evaluating efficacy; (ii) testing the compound in vivo and measuring the effect of the compound at the pain threshold; (iii) selecting a compound in which at least five potency boosts occur in step (i) and at least a twofold increase in pain threshold.

In certain embodiments, the potency boost is measured by measuring the IC ₅₀ of the compound at physiological pH and disease induced pH at least 5 measurements until the 95% confidence interval for potency boost does not change more than 15% with the addition of a new experiment. (So-called "potency boost"). In certain embodiments, the potency boost is measured at least 12 times. In another specific embodiment, the pain threshold is measured at least 12 times.

In some minor embodiments, the 95% confidence interval of the efficacy gain obtained in step (i) is at least 15%, at least 10%, at least 8%, at least 5%, at least 4%, at least 3%, or in addition to a new experiment. It does not change more than 2%.

In another incidental embodiment, the potency boost experiment of step (i) is repeated at least 5 times, at least 6 times, at least 7 times, at least 8 times, at least 9 times, or at least 10 times.

Pain thresholds can be measured in animal models of pain, in particular animal models of neuropathic pain. In one embodiment, the 95% confidence interval of the pain threshold obtained in step (ii) is at least 15%, at least 10%, at least 8%, at least 5%, at least 4%, at least 3%, or 2 with the addition of a new experiment. It does not change more than%. In certain incidental embodiments, the 95% confidence interval obtained in step (ii) is no more than 5% unchanged.

In any of the additional embodiments described above, the disease lowers the pH in the infected tissue. In certain embodiments, the disease that lowers pH in the area of infected tissue is a pain disorder, in particular neuropathic pain.

In one embodiment, identification of compounds useful for treating or preventing neuropathic pain includes: (i) a 95% confidence interval for elevated efficacy of the compound at low and physiological pH of disease induction, in cells expressing human NMDA receptors. Repeatedly evaluating at least five potency boost experiments until no further 15% change of this new experiment; (ii) Test the compound in an animal model of neuropathic pain and repeat the test at least 12 times until the 95% confidence interval did not change by more than 5% with the addition of a new experiment. Measuring; (iii) selecting at least five compounds of potency increase in step (i), and in step (ii) a compound associated with at least a two-fold increase in pain threshold.

In another embodiment, the compound exhibits a potency increase of at least 6, 7, 8, 9, 10, 15, or 20 in step (i) and at least 2, 3, 4 pain thresholds in step (ii). 5 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times increase.

Also provided are methods for alleviating the progression of an ischemic or irritant toxic cascade associated with a drop in pH by administration of a compound selected according to the methods or procedures described herein. Also provided are methods for reducing infarct volume associated with a drop in pH by administration of a compound selected according to the methods or procedures described herein. Also provided are methods for reducing cell death associated with a drop in pH by administration of a compound selected according to the methods or procedures described herein. Also provided are methods for reducing behavioral disorders associated with ischemic phenomena associated with lowering pH by administration of a compound selected according to the methods or procedures described herein.

In another aspect of the present invention, there is provided a method for treating a patient suffering from ischemic injury by administering a compound selected according to the methods or processes described herein or for preventing or treating neurotoxicity associated with ischemic injury. In addition, the following diseases or neurological conditions by the administration of a compound selected according to the methods or processes described herein include, but are not limited to: Parkinson's disease, chronic nerve damage, chronic pain syndrome, But not, for example, diabetic neuropathy, ischemia, ischemia due to transient or persistent vascular occlusion, seizures, "proliferative inhibition", hypocarbonates, hypercarbonates, diabetic ketoesidosis, prenatal lyrics, bypass surgery Cognitive impairment, vasospasm after subarachnoid hemorrhage, spinal cord injury, traumatic brain injury, overlapping epilepsy, epilepsy, hypoxia, perinatal hypoxia, (brain) concussion, migraine, hypocarbonate, hyperventilation, lactosis Provided are methods for the treatment of doss, fetal death, cerebral glyoma, and / or retinopathy during birth. In addition, compounds selected in accordance with the methods or procedures described herein may be used for patients with predisposition to ischemic phenomena such as, for example, genetic predisposition, patients with vascular spasms, or for heart bypass surgery. In patients who receive it, it can be used prophylactically to prevent or protect against such diseases or neurological conditions.

details

The present invention provides an improved method of selecting a safe and effective NMDA receptor antagonist for the treatment or prevention of pH lowering diseases in the area of infected tissue for better human clinical performance. This is done using cells expressing human NMDA receptors to assess the elevated pH potency of the ex vivo compound. The inventors surprisingly note that with respect to the methods described herein, the improved pH potency assessed in cells expressing human NMDA receptors is an improved method of selecting safe NMDA receptor antagonists, with the same results as in cells expressing other mammalian NMDA receptors. It was found that it is not shown. The methods provided herein can be used for the selection of safe NMDA receptor antagonists for the treatment or prevention of a disease in humans at which pH is lowered in the area of infected tissue, and in certain embodiments, neuropathic pain, ischemia, Parkinson's disease, Useful for diseases such as epilepsy and traumatic brain injury.

In one embodiment, in a cell expressing a human NMDA receptor, (for example, IC ₅₀ in the low pH of the IC ₅₀ / disease induction at physiological pH) efficacy of tea compounds in disease induction pH and physiologic pH of evaluating the Provided are methods for identifying compounds useful for treating or preventing a disease of which pH is lowered in an area of infected tissue. The assessment of potency boost was repeated at least 5 measurements until the 95% confidence interval for potency boost did not change by more than 15% with the addition of a new experiment, resulting in an IC ₅₀ ("potency boost" of the compound at physiological and disease induced pH). ") May be measured.

In some embodiments, the 95% confidence interval does not change by at least 10%, at least 8%, at least 5%, at least 4%, at least 3%, or at least 2% with the addition of a new experiment.

In another incidental embodiment, the potency boost experiment is repeated five times, at least six times, at least seven times, at least eight times, at least nine times, or at least ten times.

In another aspect of the invention, identification of compounds useful for treating or preventing pain disorders in an infected tissue region includes: (i) compounds that inhibit human NMDA receptors at disease-induced and physiological pH in cells expressing human NMDA receptors. Evaluating the potency of the; (ii) testing the compound in vivo and measuring the effect of the compound on the pain threshold; (iii) selecting a compound in which at least five potency boosts occur in step (i) and at least a twofold increase in pain threshold.

In another aspect of the invention, identification of compounds useful for treating or preventing a disease in an infected tissue region includes: (i) in cells expressing human NMDA receptors, increasing the potency of the compound at low and physiological pH of disease induction. Repeatedly evaluating at least five potency boost experiments until the 95% confidence interval has not changed more than 15% by the addition of a new experiment; (ii) testing the compound in an animal model of neuropathic pain and repeating the experiment at least five times until the 95% confidence interval did not change by at least 15% with the addition of a new experiment. Measuring; (iii) selecting at least five compounds of potency increase in step (i), and in step (ii) a compound associated with at least a two-fold increase in pain threshold.

In some embodiments, the 95% confidence interval obtained in step (i) is at least 15%, at least 10%, at least 8%, at least 5%, at least 4%, at least 3%, or 2 in addition to a new experiment. No more than%

In another additional embodiment, the potency boost experiment of step (i) is repeated 5 times, at least 6 times, at least 7 times, at least 8 times, at least 9 times, or at least 10 times.

In some additional embodiments, the 95% confidence interval obtained in step (ii) is at least 15%, at least 10%, at least 8%, at least 5%, at least 4%, at least 3%, or 2 in addition to a new experiment. No more than% In certain incidental embodiments, the 95% confidence interval obtained in step (ii) does not change more than 5%.

In another sub-embodiment, the efficacy boost experiment of step (ii) is five, at least six, at least seven, at least eight, at least nine, at least ten, at least eleven, at least twelve, at least thirteen Repeat at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 times. In certain incidental embodiments, the efficacy boost experiment of step (ii) is repeated at least 20 times.

In an additional embodiment of any of the embodiments described above, the disease that lowers the pH in the affected tissue area is neuropathic pain.

In one embodiment, the identification of compounds useful for treating or preventing neuropathic pain includes: (i) 95% confidence in the efficacy of the compound at low and physiological pH of disease induction, in cells expressing human NMDA receptors. Repeatedly evaluating at least five efficacy elevation experiments until the interval has not changed more than 15% by the addition of a new experiment; (ii) Test the compound in an animal model of neuropathic pain and repeat the test at least 12 times until the 95% confidence interval did not change by more than 5% with the addition of a new experiment. Measuring; (iii) selecting at least five compounds of potency increase in step (i), and in step (ii) a compound associated with at least a two-fold increase in pain threshold.

In some embodiments of any of the foregoing embodiments, the cell can express an NR1 subunit and at least one NR subunit of a human NMDA receptor. In another embodiment, the NR2 subunit can be an NR2B subunit. In other embodiments, the NR2 subunit can be an NR2A subunit.

In some additional embodiments, the physiological pH is about 7.6. In another aspect of the invention, the compounds identified by the methods described herein have increased activity in brain tissues having lower-than-normal pH because of the pathological condition. The acidic environment caused by ischemic tissue or other disease during stroke is utilized as a switch to activate the neuroprotective agents described herein. Thus, the drug becomes less active at this site, thereby minimizing side effects in uninfected tissues. Conditions that can change local pH include: hypoxia due to stroke, traumatic brain injury, extensive ischemia that may occur during cardiac surgery, hypoxia that may occur due to respiratory arrest, pre-eclampsia, spinal cord injury, epilepsy, and overlapping epilepsy , Neuropathic pain, inflammatory pain, chronic pain, vascular dementia or glyoma tumors.

In one embodiment, the IC ₅₀ value of the compound is 0.01 to 10 μM, 0.01 to 9 μM, 0.01 to 8 μM, 0.01 to 7 μM, 0.01 to 6 μM, 0.01 to 5 μM, 0.01 to 4 μM, 0.01 to 3 μM, 0.01 to 2 μM, 0.01 to 1 μM, 0.05 to 7 μM, 0.05 to 6 μM, 0.05 to 5 μM, 0.05 to 4 μM, 0.05 to 3 μM, 0.05 to 2 μM, 0.05 to 1 μM, 0.05 to 0.5 μM, 0.1 to 7 μM, 0.1 to 6 μM, 0.1 to 5 μM, 0.1 to 4 μM, 0.1 to 3 μM, 0.1 to 2 μM, 0.1 to 1 μM, 0.1 to 0.5 μM, 0.1 to 0.4 μM, 0.1 to 0.3 μM, or 0.1 to 0.2 μM.

In certain embodiments, the compounds, compared to the IC ₅₀ at physiological pH for the IC ₅₀ in the infected pH (i.e., (IC ₅₀₎ in the IC ₅₀ / Dis pH in phys pH) When, at least two, at least three, Efficacy elevation of at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, or at least 20.

In one embodiment, the compound has an IC ₅₀ value of less than 10 μM at a pH of about 6-9. In one embodiment, the compound has an IC ₅₀ value of less than 10 μM at a pH of about 6.9. In another embodiment, the compound has an IC ₅₀ value of less than 10 μM at a pH of about 7.6. In one embodiment, the compound has an IC ₅₀ value of less than 10 μM at physiological pH. In one embodiment, the compound has an IC ₅₀ value of less than 10 μM at infected pH.

In one embodiment, the IC ₅₀ value of the compound at pH 6.9 is 0.01 to 10 μM, 0.01 to 9 μM, 0.01 to 8 μM, 0.01 to 7 μM, 0.01 to 6 μM, 0.01 to 5 μM, 0.01 to 4 μM, 0.01 to 3 μM, 0.01 to 2 μM, 0.01 to 1 μM, 0.05 to 7 μM, 0.05 to 6 μM, 0.05 to 5 μM, 0.05 to 4 μM, 0.05 to 3 μM, 0.05 to 2 μM, 0.05 to 1 μM, 0.05 to 0.5 μM, 0.1 to 7 μM, 0.1 to 6 μM, 0.1 to 5 μM, 0.1 to 4 μM, 0.1 to 3 μM, 0.1 to 2 μM, 0.1 to 1 μM, 0.1 to 0.5 μM, 0.1 to 0.4 μM, 0.1 to 0.3 μM, or 0.1 to 0.2 μM, and the ratio of IC ₅₀ values at pH 7.6 to pH 6.9 for the compound is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, Greater than 20, 25, 30, 40, 50, 60, 70, 80, 90, or 100.

In one embodiment, the IC ₅₀ value of the compound at pH 6.9 is 0.01 to 10 μM, 0.01 to 9 μM, 0.01 to 8 μM, 0.01 to 7 μM, 0.01 to 6 μM, 0.01 to 5 μM, 0.01 to 4 μM. , 0.01 to 3 μM, 0.01 to 2 μM, 0.01 to 1 μM, 0.05 to 7 μM, 0.05 to 6 μM, 0.05 to 5 μM, 0.05 to 4 μM, 0.05 to 3 μM, 0.05 to 2 μM, 0.05 to 1 μM , 0.05 to 0.5 μM, 0.1 to 7 μM, 0.1 to 6 μM, 0.1 to 5 μM, 0.1 to 4 μM, 0.1 to 3 μM, 0.1 to 2 μM, 0.1 to 1 μM, 0.1 to 0.5 μM, 0.1 to 0.4 μM , 0.1 to 0.3 μM, or 0.1 to 0.2 μM, and the ratio of the IC ₅₀ value at pH 7.6 to pH 6.9 for the compound is 1 to 100, 2 to 100, 3 to 100, 4 to 100, 5 to 100, 6 To 100, 7 to 100, 8 to 100, 9 to 100, 10 to 100, 15 to 100, 20 to 100, 25 to 100, 30 to 100, 40 to 100, 50 to 100, 60 to 100, 70 to 100 , 80 to 100, or 90 To 100.

In another embodiment, the IC ₅₀ value of the compound at a pH of about 7.6 is 0.01 to 10 μM, 0.01 to 9 μM, 0.01 to 8 μM, 0.01 to 7 μM, 0.01 to 6 μM, 0.01 to 5 μM, 0.01 to 4 μM, 0.01 to 3 μM, 0.01 to 2 μM, 0.01 to 1 μM, 0.05 to 7 μM, 0.05 to 6 μM, 0.05 to 5 μM, 0.05 to 4 μM, 0.05 to 3 μM, 0.05 to 2 μM, 0.05 to 1 μM, 0.05 to 0.5 μM, 0.1 to 7 μM, 0.1 to 6 μM, 0.1 to 5 μM, 0.1 to 4 μM, 0.1 to 3 μM, 0.1 to 2 μM, 0.1 to 1 μM, 0.1 to 0.5 μM, 0.1 to 0.4 μM, 0.1-0.3 μM, or 0.1-0.2 μM. In certain such embodiments, the compound is, at pH 7.6 to pH 6.9, 1 to 100, 2 to 100, 3 to 100, 4 to 100, 5 to 100, 6 to 100, 7 to 100, 8 to 100 , 9-100, 10-100, 15-100, 20-100, 25-100, 30-100, 40-100, 50-100, 60-100, 70-100, 80-100, or 90-100 The ratio of IC ₅₀ values is shown. In another specific embodiment, the compound exhibits a ratio of less than 10, or less than 5, or 4, 3, 2 or 1.

In another embodiment, testing in cells expressing human NMDA receptors, assaying the potency increase of the compound by comparing the potency at physiological pH against “disease induced low pH” (eg, IC ₅₀ / at phys pH). Provides a method of selecting compounds that exhibit at least a 5 increase in potency as determined by an experiment with IC ₅₀ ) at disease induced low pH, until the 95% confidence interval does not change by more than 15% with the addition of a new experiment. Repeat at least 12 experiments. In another preferred embodiment, there is provided a method of selecting a compound, or in an animal model of neuropathic pain, determined by repeating at least 15 experiments until the 95% confidence interval has not changed by at least 15% by the addition of a new experiment. Compounds are selected which increase the measured pain threshold by at least twofold. In another particular embodiment, “disease induced low pH” may be associated with an ischemic disease such as stroke.

In one embodiment, the compound selected according to the methods and processes described herein is a selective NR1 / NR2A human NMDA receptor and / or NR1 / NR2B human NMDA receptor antagonist. In one embodiment, the compound is not an NMDA receptor channel blocker.

In a further embodiment, the compound does not exhibit substantial toxic side effects such as motor impairment, cognitive impairment and cardiac toxicity. Additionally or alternatively, the compound has a therapeutic index greater than or equal to at least 2: 1. In a further or alternative embodiment, the compound selectively binds at least 10-fold to the NMDA receptor over any other glutamate receptor. In one embodiment, oocytes are used to measure potency boost. In another embodiment, the middle cerebral artery occlusion model is used as an animal model of transient focal ischemia, for example, a rodent animal model such as a mouse.

In another embodiment, the compound has at least 6, 7, 8, 9, 10, 15, or 20 increased efficacy in step (i), and at least 2, 3 times the pain threshold of step (ii). , 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times increase.

Also provided is a method of alleviating the progression of an ischemic or excitotoxic cascade associated with a drop in pH by administration of a compound selected according to the methods or procedures described herein. In addition, methods of reducing infarct volume associated with a drop in pH by administration of a compound selected according to the methods or processes described herein are provided. Also provided are methods for reducing cell death associated with a drop in pH by administration of a compound selected according to the methods or procedures described herein. Also provided is a method of reducing behavioral deficits associated with ischemic phenomena associated with a drop in pH by administration of a compound selected according to the methods or processes described herein.

In a further aspect of the invention, there is provided a method for treating ischemic injury in a patient or for treating or preventing neuronal toxicity associated with ischemic injury by administering a compound selected according to the methods or processes described herein. . In addition, the following diseases or neurological conditions by the administration of a compound selected according to the methods or processes described herein include, but are not limited to: Parkinson's disease, chronic nerve damage, chronic pain syndrome, But not, for example, diabetic neuropathy, ischemia, ischemia due to transient or persistent vascular occlusion, seizures, "proliferative inhibition", hypocarbonates, hypercarbonates, diabetic ketoesidosis, prenatal lyrics, bypass surgery Cognitive impairment, vasospasm after subarachnoid hemorrhage, spinal cord injury, traumatic brain injury, overlapping epilepsy, epilepsy, hypoxia, perinatal hypoxia, (brain) concussion, migraine, hypocarbonate, hyperventilation, lactosis, fetal birth , Brain glyomas, and / or retinopathy, and the like. In addition, the compounds selected according to the methods or processes described herein are, for example, in patients with predisposition to ischemic phenomena such as hereditary predisposition, or in patients with vascular spasm, or in patients undergoing heart bypass surgery. It can be used prophylactically to prevent or protect against such diseases or neurological conditions.

Analysis of Efficacy Elevation

The term "oocyte" is the final product of oogenesis and also refers to the egg of an adult animal which is an oocyte, primary oocyte, secondary oocyte, respectively.

"Transfection" refers to the introduction of DNA into a host cell. Cells do not naturally absorb DNA. Thus, various technical “tricks” are used to facilitate gene transfer. Many methods of transfection, such as CaPO ₄ method, lipid-based method, and electroporation, are known to those skilled in the art (J. Sambrook, E. Fritsch, T. Maniatis). Molecular Cloning: A Laboratory Manual, Cold Spring Laboratory Press, 1989).

Expression of NMDA Receptor in Cells

In a main aspect of the invention, the potency boost of a compound is determined in cells expressing at least one human NMDA receptor. In one embodiment, the cell may endogenously express human NMDA receptors. Cells capable of endogenously expressing the NMDA receptor include: hepatocytes, P19 cells, neuroepithelial cells, neuroendothelial cells, dopaminergic black matter neurons, astroglia, macrophage neuroendocrine cells, chromosomal neurons, cerebellar neurons, brain stem Cells, hepatic brain neurons, midbrain neurons, posterior brain neurons, spinal cord neurons, spinal cord neurons, olfactory neurons, cortical neurons, cerebellar granule cells, hippocampal neurons, diaphragm neurons, caudal cells, cortical cells, striatum, posterior cells, thalamus cells, CA1 pyramidal cells, cerebral basal ganglia cells, layer IV neurons, somatosensory cortical neurons, oocytes, placental cells and pancreatic cells, and the like.

In another embodiment, the cell can be genetically modified to express human NMDA receptors. In one specific embodiment, oocytes may be genetically modified to express human NMDA receptors. Any suitable oocyte may be used by those skilled in the art, including but not limited to frog oocytes, such as Xenopus oocytes , including Xenopus laevis, Xenopus tropicalis, Xenopus muelleri, Xenopus wittei, Xenopus gilli , And Xenopus borealis , including but not limited to. In one embodiment, the oocyte can be isolated from the ovary of the animal by any technique known to those skilled in the art.

In another embodiment, any suitable cell type, including primary cell lines, may be genetically modified to express human NMDA receptors, including: Chinese hamster (CHO) oocytes, HEK renal cells, bacterial cells, E. coli cells, yeast cells, neurons, heart cells, lung cells, gastric cells, splenocytes, pancreatic cells, renal cells, hepatocytes, enterocytes, skin cells, hair cells, hypothalamus cells, pituitary cells, epithelial cells, Fibroblasts, nervous system cells, keratinocytes, hematopoietic cells, melanocytes, chondrocytes, lymphocytes (B and T), macrophages, monosites, mononuclear cells, cardiomyocytes, other myocytes, oocytes, epidermal cells, endothelial cells , Langerhan's eyelet cells, blood cells, blood precursor cells, bone cells, bone precursor cells, neural stem cells, primordial stem cells, hepatocytes, keratinocytes, umbilical vein endothelial cells, aortic endothelial cells, capillaries Blood cells, fibroblasts, hepatic stellate cells, aortic smooth muscle cells, cardiomyocytes, neurons, Schiffer cells, smooth muscle cells, Schwann cells, and epithelial cells, erythrosites, platelets, neutrophils, lymphocytes, eosinophils, basophils, adiposites , Chondroitin, pancreatic islet cells, senile gland cells, parathyroid cells, parotid cells, tumor cells, somatic cells, pituitary cells, adrenal cells, bubble cells, renal cells, retinal cells, hepatocytes, corn cells, heart cells, Pacemaker cells, splenocytes, antigen presenting cells, memory cells, T cells, B cells, plasma cells, myocytes, ovary cells, uterine cells, prostate cells, vaginal epithelial cells, sperm cells, testes cells, germ cells, egg cells, ray Dict cells, peritubler cells, sertoli cells, lutein cells, cervical cells, endometrial cells, mammary cells, follicle cells, mucous cells, ciliated cells, non-keratin epithelial cells, keratinous epithelial cells, lung cells, germ cells , Although the like columnar epithelial cells, squamous epithelial cells, liver cell times, austenite five sites, five blasting austenite, and the austenite Eau last, not limited to this.

In another embodiment, the cell can be genetically modified to express the selected human NMDA receptor subunit. NMDA receptors consist of NR1, NR2 (A, B, C, and D), and NR3 (A and B) subunits, which determine the functional properties of the native NMDA receptor. NMDA receptors are heteromeric proteins composed of NR1, NR2 and / or NR3. The DNA encoding of any NMDA receptor subunit from humans can be used to genetically modify the cell. Table 1 provides GenEMBL accession numbers for human NMDA receptor subunits.

For example, mRNA can be synthesized from cDNA templates and injected into cells. Alternatively, the cDNA encoding the receptor subunit may be inserted into a construct or vector prior to being inserted into the cell. Techniques that can be used to introduce DNA constructs or vectors into host cells include calcium phosphate / DNA co-precipitation, microinjection of DNA into the nucleus, electroporation, and intact cells. Bacterial plasma fusion, transfection, or other methods known to those skilled in the art. The DNA can be linear or circular, relaxed or supercoiled DNA. Various techniques for introduction into mammalian cells are described, for example, in Keown et al., Methods in Enzymology Vol. 185, pp. 527-537 (1990).

Constructs or vectors can be prepared according to methods known in the art. Constructs can be prepared using bacterial vectors, including prokaryotic replication systems (eg, origin recognition at each step by E. coli), and can be replicated and analyzed. It is also possible to use selectable markers. Once the vector containing the construct is completed, it can be further facilitated by such things as bacterial sequences, linearization, elimination of the introduction of short deletions in homologous sequences. After the last manipulation, the construct can be introduced into the cell.

The present invention also encompasses recombinant constructs comprising one or more of the sequences described above. The construct may be in the form of a vector, such as a plasmid or a viral vector, which can be inserted into the sequences of the present invention, in a forward or reverse orientation. Also, the construct may include a regulatory sequence that includes a promoter linked to be operable to the sequence, for example. Many of suitable vectors and promoters are known to those skilled in the art and are commercially available. The following vectors can be provided by way of example: pBs, pQE-9 (Qiagen), phagescripts, PsiX174, pBluescript SK, pBsKS, pBSSK, pGEM, pNH8a, pNH16a, pNH18a, pNH46a (Stratagene); pTrc99A, pKK223-3, pKK233-3, pDR540, pRIT5 (Pharmacia). Eukaryotic: pCiNeo, pWLneo, pSv2cat, pOG44, pXTl, pSG (Stratagene) pSVK3, pBPv, pMSG, pSVL (Pharmiacia). In addition, other plasmids and vectors can be used as long as they are replicable and viable in the host. Vectors known in the art and commercially available ones (and variants or derivatives thereof) can be used according to the invention designed to have one or more recombinant sites for use in the methods of the invention. Such vectors can be obtained, for example, from Vector Laboratories Inc., Invitrogen, Promega, Novagen, NEB, Clontech, Boehringer Mannheim, Pharmacia, EpiCenter, OriGenes Technologies Inc., Stratagene, PerkinElmer, Pharmingen, and Research Genetics. Other interesting vectors include eukaryotic expression vectors such as pFastBac, pFastBacHT, pFastBacDUAL, pSFV, and pTet-Splice (Invitrogen), pEUK-Cl, pPUR, pMAM, pMAMneo, pBHOl, pBI121, pDR2, pCMVEBNA, and pYACneo (Clontech), pSVK3, pSVL, pMSG, pCHl lO, and pKK232-8 (Pharmacia, Inc.), p3'SS, pXTl, pSG5, pPbac, pMbac, pMClneo, and pOG44 (Stratagene, Inc.), and pYES2 , pAC360, pBlueBacHis A, B, and C, pVL1392, pBlueBacIII, pCDM8, pcDNAl, pZeoSV, pcDNA3 pREP4, pCEP4, and pEBVHis (Invitrogen, Corp.) and variants or derivatives thereof.

Additional vectors suitable for use in the present invention include pUC18, pUC19, pBlueScript, pSPORT, cosmid, phagemid, YACs (yeast artificial chromosome), BACs (bacterial artificial chromosome), Pl (E. coli phage), pQE70, pQE60 , pQE9 (quagen), pBS vector, PhageScript vector, BlueScript vector, pNH8A, pNH16A, pNH18A, pNH46A (Stratagene), pcDNA3 (Invitrogen), pGEX, pTrsfus, pTrc99A, pET-5, pET-9, pKK223-3, pKK233-3, pDR540, pRIT5 (Pharmacia), pSPORTl, pSPORT2, pCMVSPORT2.0 and pSV-SPORTl (Invitrogen) and variants or derivatives thereof. Viral vectors may also use such as lentiviral vectors (see, eg, WO 03/059923; Tiscornia et al. PNAS 100: 1844-1848 (2003)). Additionally interesting vectors include pTrxFus, pThioHis, pLEX, pTrcHis, pTrcHis2, pRSET, pBlueBacHis2, pcDNA3. I / His, pcDNA3.1 (-) / Myc-His, pSecTag, pEBVHis, pPIC9K, pPIC3.5K, pAO815, pPICZ, pPICZA, pPICZB, pPICZC, pGAPZA, pGAPZB, pGAPZC, pBlueBacPac2, pBlueBacPac2 , pSinHis, pIND, pIND (SPl), pVgRXR, pcDNA2.1, pYES2, pZErO1.1, pZEiO-2.1, pCR-Blunt, pSE280, pSE380, pSE420, pVL1392, pVL1393, pCDM8, pcDNA1.1, pcDNA1.1 / Amp, pcDNA3.1, pcDNA3.1 / Zeo, pSe, SV2, pRc / CMV2, pRc / RSV, pREP4, pREP7, pREP8, pREP9, pREP 10, pCEP4, pEBVHis, pCR3.1, pCR2.1, pCR3.1 -Uni, and pCRBac (Invitrogen); λ ExCell, λ gt11, pTrc99A, pKK223-3, pGEX-1 λ T, pGEX-2T, pGEX-2TK, pGEX-4T-1, pGEX-4T-2, pGEX-4T-3, pGEX-3X, pGEX- 5X-1, pGEX-5X-2, pGEX-5X-3, pEZZ18, pRIT2T, pMC1871, pSVK3, pSVL, pMSG, pCHHO, pKK232-8, pSL1180, pNEO, and pUC4K (Pharmacia); pSCREEN-1b (+), pT7Blue (R), pT7Blue-2, pCITE-4abc (+), pOCUS-2, pTAg, pET-32LIC, pET-30LIC, pBAC-2cp LIC, pBACgus-2cp LIC, pT7Blue-2 LIC, pT7Blue-2, λ SCREEN-1, λ BlueSTAR, pET-3abcd, pET-7abc, pET9abcd, pET11abcd, pET12abc, pET-14b, pET-15b, pET-16b, pET-17b-pET-17xb, pET- 19b, pET-20b (+), pET-21abcd (+), pET-22b (+), pET-23abcd (+), pET-24abcd (+), pET-25b (+), pET-26b (+) , pET-27b (+), pET-28abc (+), pET-29abc (+), pET-30abc (+), pET-31b (+), pET-32abc (+), pET-33b (+), pBAC-1, pBACgus-1, pBAC4x-l, pBACgus4x-1, pBAC-3cp, pBACgus-2cp, pBACsurf-1, plg, Signal plg, pYX, Selecta Vecta-Neo, Selecta Vecta-Hyg, and Selecta Vecta-Gpt Novagen; pLexA, pB42AD, pGBT9, pAS2-1, pGAD424, pACT2, pGAD GL, pGAD GH, pGADIO, pGilda, pEZM3, pEGFP, pEGFP-1, pEGFP-N, pEGFP-C, pEBFP, pGFPuv, pGFP, p6xHis-G pSEAP2-Basic, pSEAP2-Contral, pSEAP2-Promoter, pSEAP2-Enhancer, pβgal-Basic, pβgal-Control, pβgal-Promoter, pβgal-Enhancer, pCMV, pTet-Off, pTet-On, pTK-Hyg, pRetro-Off, pRetro-On, pIRESlneo, pIRESlnyg, pLXSN, pLNCX, pLAPSN, pMAMneo, pMAMneo-CAT, pMAMneo-LUC, pPUR, pSV2neo, pYEX4T-1 / 2/3, pYEX-S1, pBacPAK-His, pBacPAK8W31 BacPAK6, pTriplEx, λgt10, λgt11, pWE15, and λTripffix (Clontech); Lambda ZAP II, pBK-CMV, pBK-RSV, pBluescript II KS +/-, pBluescript II SK +/-, pAD-GAL4, pBD-GAL4 Cam, pSurfscript, Lambda FIX II, Lambda DASH, Lambda EMBL3, Lambda EMBL4, SuperCos, pCR-Scrigt Amp, pCR-Script Cam, pCR-Script Direct, pBS +/-, pBC KS +/-, pBC SK +/-, Phagescript, pCAL-n-EK, pCAL-n, pCAL-c, pCAL-kc, pET-3abcd, pET-11abcd, pSPUTK, pESP-1, pCMVLacI, pOPRSVI / MCS, pOPI3 CAT, pXT1, pSG5, pPbac, pMbac, pMClneo, pMClneo Poly A, pOG44, pOG45, pFRTβGAL, pNERTβGAL , pRS404, pRS405, pRS406, pRS413, pRS414, pRS415, and pRS416 (Stratagene).

Additional vectors include, for example, pPC86, pDBLeu, pDBTrp, pPC97, p2.5, pGADl-3, pGADIO, pACt, pACT2, pGADGL, pGADGH, pAS2-l, pGAD424, pGBT8, pGBT9, pGAD-GAL4, pLexA, pBD-GAL4, pHISi, pHISi-1, placZi, pB42AD, pDG202, pJK202, pJG4-5, pNLexA, pYESTrp and variants or derivatives thereof.

In addition, selectable markers may be inserted into the vector to allow selection of cells containing human NMDA receptor subunits. Suitable selectable markers include: genes that confer the ability to grow on specific media substrates, such as the tk gene (thymidine kinase) conferring the ability to grow on HAT medium (hypoxanthine, aminopterin and thymidine). Or hprt gene (hypoxanthine phosphoribosyltransferase); Bacterial gpt genes (guanine / xanthine phosphoribosyltransferase) that can grow in MAX medium (mycophenolic acid, adenine, and xanthine). See, eg, Song, KY., Et al. Proc. Natl Acad. Sci. USA 84: 6820-6824 (1987); See Sambrook, J., et al., Molecular Cloning-A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1989), Chapter 16. Selectable markers include: genes that provide resistance to compounds such as antibiotics, genes that give them the ability to grow on selected substrates, detectable signals such as green fluorescent protein, enhanced green fluorescent protein (eGFT) and Genes encoding proteins that exhibit the same luminescence or fluorescence. A wide range of such markers are known and available and include, for example, antibiotic resistance genes such as neomycin resistance gene (neo) (Southern, P., and P. Berg, J. MoI. Appl. Genet. 1: 327-341 (1982)); And hygromycin resistance gene (hyg) (Nucleic Acids Research U -.6895-69 U (1983), and Te Riele, H., et al, Nature 348: 649-651 (1990)). Other selectable marker genes include: acetohydroxy acid synthase (AHAS), alkaline phosphatase (AP), beta galactosidase (LacZ), beta glucoronidase (GUS), chloramphenicol acetyltransferase ( CAT), green fluorescent protein (GFP), red fluorescent protein (RFP), yellow fluorescent protein (YFP), cyan fluorescent protein (CFP), horseradish peroxidase (HRP), luciferase (Luc), nopalin syntha Azedes, octopine synthase (OCS), and derivatives thereof. Multiselectable markers are commonly used to confer resistance to ampicillin, bleomycin, chloramphenicol, gentamycin, hygromycin, kanamycin, lincomycin, methotrexate, porfinoricin, puromycin, and tetracycline.

Methods of combining antibiotic resistance genes with negative selection factors are well known to those of skill in the art (eg, WO 99/15650; US Patent No. 6,080,576; US Patent No. 6.136,566; Niwa, et al., J. Biochem 113: 343-349 (1993) and Yoshida, et al., Transgenic Research, 4: 277-287 (1995).

Cells successfully transformed to express human NMDA receptors can be identified through functional or molecular analysis. In one embodiment, cells, such as oocytes into which the human NMDA receptor subunit cRNA is inserted, can be tested via electrophysiological recordings for the presence of functional human NMDA receptors. In another embodiment, the cells into which the DNA encoding the human NMDA receptor subunit gene and the selectable marker gene are inserted can be grown in an appropriately selected medium to identify cells that are appropriately integrated. Cells exhibiting the desired phenotype can then be analyzed by confinement analysis, electrophoresis, Southern analysis, polymerase chain reaction, or other techniques known to those skilled in the art. By identifying fragments that show appropriate insertion into the target gene site, one can identify whether homologous recombination occurs in the cell to inactivate or modify the target gene.

Potency Boost Experiment

In a further aspect of the invention, cells expressing human NMDA receptors can be used to determine the potency boost of certain compounds, such as those described according to the methods and procedures herein.

 Efficacy of the compound was repeated for the effect of the compound at disease-induced low and physiological pH in cells expressing human NMDA receptors, repeating the efficacy-elevation experiment until the 95% confidence interval did not change more than 15% with the addition of a new experiment. This can be determined by testing. In one preferred embodiment, experiments to elevate the efficacy of compounds evaluated at disease-induced low and physiological pH in cells expressing human NMDA receptors are performed until the 95% confidence interval does not change more than 15% with the addition of a new experiment. By repeating at least five potency boost experiments, a method is provided for selecting compounds in which the potency boost of the compounds determined in the experiment exhibits a potency boost of at least five.

"Disease-induced low pH" is defined as a drop in pH associated with any disease or condition referred to herein. The "disease induced low pH" is about 6.4 to 7.1 and is generally about 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, or 7.1. Physiological brain tissue pH is about 7.2 to about 7.8, and generally about 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, or 7.8. In one embodiment, the "disease induced low pH" may be associated with an ischemic disease such as stroke and the like.

In one embodiment, the disease induced low pH is about 6.9. In another embodiment, the disease induced low pH is about 6.7 to about 7.1.

In one embodiment, the physiological pH is about 7.6. In another embodiment, the physiological pH is about 7.4 to 7.8.

“Efficacy boost” experiments are conducted at concentrations of compounds that cause half-maximal inhibition of NMDA channel action on a compound, at physiological pH, such as pH 7.6 and ischemic or neuropathic pain pH, such as pH 6.9. Determine (IC ₅₀ ). Any method known in the art can be used to determine the IC ₅₀ value for a compound. IC ₅₀ values are expressed as percentages and can be averaged together to determine the average shift in IC ₅₀ . In one embodiment, two electrode voltage-clamp recording can be used to determine the IC ₅₀ value for a compound. The glass microelectrode can be filled with potassium chloride such that the voltage electrode contains a lower concentration of potassium chloride than the current electrode. The cells can be placed in a chamber and physiological solution can be added. External pH may be adjusted to ischemia or neuropathic pain pH, such as pH 6.9 or physiological pH, such as pH 7.6. The most effective concentrations of glutamate and glycine can then be applied in a continuous manner, followed by addition of various concentrations of test compound to glutamate / glycine to obtain a dose response curve. The level of inhibition by the applied antagonist can be expressed as a percentage of the initial glutamate response. These values can be aggregated and averaged across cells, for example across oocytes from a single frog. The average percent response at each antagonist concentration can be formulated as (100-min) / (1 + ([conc] / IC ₅₀ ) ^nH ) + min, where min is the residual percent response in saturated antagonists, IC ₅₀ is the concentration of the antagonist that causes half of the maximum achievable inhibition, and nH is the slope factor indicating the steepness of the inhibition curve. min can be defined as equal to or greater than zero. For example, min can be set to zero in experiments with known channel blockers. IC ₅₀ values obtained at physiological pH and ischemic pH are expressed as percentages and can be averaged in aggregate to determine the average shift in IC ₅₀ . In a further embodiment, the compound is at least 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 at disease induced low and physiological pH. Efficacy gains greater than, 22 or 23.

Efficacy boost experiments can be repeated until the 95% confidence interval has not changed more than 15% with the addition of a new experiment. In another embodiment, the potency boost experiment has a 95% confidence interval of about 14%, 13,%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5 with the addition of a new experiment. Repeat until no more than%, 4%, 3%, or 2%. In a further embodiment, the potency boost experiment has a 96%, 97%, 98% or 99% confidence interval of about 10%, 9%, 8%, 7%, 6%, 5%, 4% with the addition of a new experiment. Repeat until no more than 3% or 2% has not changed.

Animal model

In one aspect of the invention, (i) efficacy of increasing the compound's efficacy at disease-induced low and physiological pH in cells expressing human NMDA receptors until the 95% confidence interval does not change by more than 15% with the addition of a new experiment. Evaluating the synergy experiment repeatedly; (ii) testing the compound in an animal model of neuropathic pain and repeating the experiment until the 95% confidence interval does not change more than 5% with the addition of a new experiment, measuring the effect of the compound on the increase in pain threshold ; (iii) selecting a compound associated with an increase in efficacy of at least 5 in step (i) and at least a 2 fold increase in pain threshold in step (ii). Methods of identifying chemical compounds useful for treating or preventing are provided. In one embodiment, the candidate drug must meet or exceed ex vivo and in vivo criteria to be a good drug for human use.

In a preferred embodiment, the pain threshold in the animal model of neuropathic pain is increased by at least a 2-fold increase in pain threshold by repeating at least 15 experiments until the 95% confidence interval does not change by at least 15% with the addition of a new experiment. It provides a compound or a method for selecting a compound selected to represent.

Animal models of neuropathic pain

In one aspect of the invention, the compounds disclosed herein may be useful for the treatment or prevention of pain, particularly neuropathic pain and related diseases.

In one aspect of the invention, (i) increasing the potency of a compound at "disease induced pH" and physiological pH in a cell, at least five potency boost experiments until the 95% confidence interval does not change more than 15% with the addition of a new experiment step of the evaluation (e.g., IC ₅₀ / IC ₅₀ in the "disease induced pH" at physiological pH) repeatedly; (ii) Test the compound in an animal model of neuropathic pain and repeat the test at least 12 times until the 95% confidence interval did not change by more than 5% with the addition of a new experiment. Measuring; (iii) a chemical compound useful for treating neuropathic pain in a mammal, in particular a human, by selecting a compound in which at least five potency elevations occur in step (i) and at least a twofold increase in pain threshold in step (ii) Provides a way to identify them.

In certain embodiments, candidate drugs must meet or exceed ex vivo and in vivo criteria in order to be good drugs for human use. In one embodiment, potency enhancement can be determined in cells that express a glutamate receptor derived from a human. In another embodiment, potency elevation can be determined in cells expressing at least one human derived NMDA, AMPA, and / or kinate receptor. In one embodiment, the cell may express an NR1 subunit and at least one NR2 subunit of an NMDA receptor. In further embodiments, the NR2 subunit may be an NR2B subunit. In another embodiment, the NR2 subunit may be an NR2A subunit.

In a more general aspect of the invention, (i) an experiment exhibits at least 5 potency boosts, wherein the potency boost of the compound is such that a "disease induced pH" and a potency boost of the compound at physiological pH (eg, physiological the IC ₅₀₎ in the IC ₅₀ / "disease induced pH" in pH, 95% by repeating at least five times the efficacy increases experiment until the confidence interval does not change more than 15% with the addition of a new experiment, the test in the cell, (ii ) Test the compound in an animal model of neuropathic pain and measure the effect of the compound on the increase in pain threshold at least 12 experiments repeatedly until the 95% confidence interval does not change more than 5% with the addition of a new experiment. (Iii) reducing the pH in a manner that activates the NMDA receptor antagonist, from selecting a compound in which at least five potency elevations occur in step (i) and at least a twofold increase in pain threshold in step (ii) The compound for treating the disease is selected, the method is provided. In one embodiment, potency elevation can be determined in cells expressing glutamate receptors. In another embodiment, the potency boost can be determined in cells expressing NMDA, AMPA, and / or kinate receptors. In one embodiment, the cell may express an NR1 subunit and at least one NR2 subunit of an NMDA receptor. In further embodiments, the NR2 subunit may be an NR2B subunit. In another embodiment, the NR2 subunit may be an NR2A subunit.

In one embodiment, the animal model of neuropathic pain may be selected from the group including, but not limited to, chronic stenosis injury model, partial sciatic ligation model, spinal cord ligation model, or other models known to those skilled in the art. In certain embodiments, the spinal cord nerve ligation model can be used as an in vivo animal model.

The “pain threshold” is a measure of the amount of stimulation needed to experience a sense of pain. In chronic neuropathic pain animal models, animals are injured and chronic pain conditions are induced. Thereafter, noxious stimulus can be applied and the amount of time an animal can withstand it without responding to the stimulus can be calculated. For example, an intact animal may be exposed to a cold surface for 20 minutes until its pore is removed from the surface, but only after one minute of damage, such as after a neuropathic pain described below You can also reap. Examples of noxious stimuli include, but are not limited to, heat, cold, mechanical and chemical stimuli such as von Frey stimuli, and the like.

In one embodiment, the chronic stenosis injury model (CCI, or Bennett model) can use an animal model of neuropathic pain (eg, Bennett, Gary J. et al. Pain, 1988, 33, 87-107). Reference). In this model, the sciatic nerve of an animal (eg, a rat) can be deliberately infused in a manner that can induce the syndrome reported by a group of human patients with neuropathic pain. In particular, the sciatic nerve may be exposed in the midthigh of the femur in the popliteal fossa proximal to the nerve. At that location, the nerve trajectory of about 7 mm can be eliminated from the attachment of the four ligatures with the tissue loosely attached around it at about 1 mm intervals. For each animal, the same dissection can be performed on the opposite side without ligation so that each animal serves as its own control. In the ligated aspect, the infected hintpo skin is clearly the source of hyperalgesia and allodynic (ie, experiencing pain resulting from stimulation that generally does not induce a pain response), and possibly spontaneous pain. For the hyperalgesia test, an invasive stimulus such as heat can be applied to the planter hint Poe from under the glass floor, and the latency (marker for pain threshold) measured with withdrawal. Responses to aspects of nerve damage tend to exhibit abnormal magnitude and persistence, excessive, for example, 30 seconds of pore elevation, and may be accompanied by prolonged licking. A typical reaction would be that the animal would only lift the poe and last less than two. For testing for cold allodynia, the animal group can be placed on a metal floor, for example, cooled at 4 ° C. For non-ligated poes, the floor does not cause pain even after 20 minutes of contact. The ligated rats can be measured for departure of neuronal damage pore, which can, for example, increase by at least 5 times, measure persistence, and increase by, for example, at least 2 times. Using this model, pain thresholds can be calculated without the drug and after administration of the compounds described herein.

In another embodiment, a partial sciatic ligation model (Seltzer model) can be used to test neuropathic pain thresholds (see Seltzer, A. et al. Pain, 1990, 43, 205-218). In this model, half of the sciatic nerve of the upper thigh of an animal, such as a rat, can be ligated in one side. Within a few hours after the operation, and for several months thereafter, the animal may develop and occasionally lick a protective action of the ipsilateral hintpo, suggesting the possibility of spontaneous pain. The planter surface of the foot may be hyperesthetic equally to harmless and noxious stimuli. Conventional measurements of noxious stimuli can be measured in animals, with exposure and no exposure to the compounds of the present invention. Hazardous stimuli may include von Frey hair stimulation, CO ₂ laser thermal vibration and pin prock. In response to repeated Bonfrey hair crust on the planter side, the exit threshold can be sharply reduced. After this series of stimuli in an engineered aspect, light touch induces an aversive response that suggests allodynia to the touch. In addition, the departure threshold for CO ₂ laser thermal vibration is significantly lowered. Threshold hazards Thermal vibrations induce a unilaterally pronounced response, suggesting thermal hyperalgesia. In addition, pin-frick can also cause this pronounced reaction (mechanical hyperalgesia). Using this model, pain thresholds can be calculated without the drug and after administration of the compounds described herein.

In another embodiment, a spinal nerve ligation model can be used to measure neuropathic pain (Kim, SH and Chung, JM Neurosci. Lett. 1991, 134, 131-134; Kim, SH and Chung, JM Pain, 1992, 50, 355-363). In this model, the L5 (or L5 + L6) spinal cord nerves are tightly ligated and then cut. Surgical procedures allow long-lasting hyperalgesia to the harmful heat and mechanical allodynia of the infected foot. The mechanical sensitivity of the infected hintpore can be measured. The bone filament is evidenced by increased foot departure from harmless mechanical stimuli applied to the hint Poe, which can rise significantly from the first day after surgical manipulation. In addition, a behavioral indication of the presence of spontaneous pain in the infected foot is shown. Such measurements can be determined by administration and non-administration of the compounds of the present invention and the pain threshold can be calculated.

After testing the compound in an animal model of neuropathic pain and measuring the effect of the compound on the pain threshold, the compound may be selected to exhibit at least a 2-fold increase in the pain threshold. In another embodiment, the compound may exhibit a minimum 3, 4, 5, 6, 7, 8, 9, 10, 15, 20 or 30 fold increase in pain threshold. In a further embodiment, the experiment can be repeated at least 15 times until the 95% confidence interval does not change by more than 10% with the addition of a new experiment. The neuropathic pain experiment can be repeated until the 95% confidence interval has not changed more than 10% with the addition of a new experiment. In another embodiment, the neuropathic pain experiment is performed until the 95% confidence interval does not change by at least about 9%, 8%, 7%, 6%, 5%, 4%, 3%, or 2% with the addition of a new experiment. Can be repeated In a further embodiment, the neuropathic pain experiment has a 96%, 97%, 98% or 99% confidence interval of about 10%, 9%, 8%, 7%, 6%, 5%, 4 with the addition of a new experiment. Repeat until no more than%, 3%, or 2%.

Another animal model of neuropathic pain is the spared neuronal damage model (see Decosterd & Woolf. Pain. 2000 Aug; 87 (2): 149-58), local inflammation of the sciatic nerve in Frank Trauma's deficiency. , And / or sciatic inflammatory neuropathy (SIN) (Aley et al Neurosci 1996; 73: 259-65) induced in peripheral nerve models of pain induced by injection of chemotherapeutic agent Vincristine. . Additional models are known to those skilled in the art. See also Zimmerman M. Eur J Pharmacol 2001; 429: 23-37; Shir et al Neurosci Lett 1990; l 15: 62-7. Wall et al Pain 1979; 7: 103-ll; DeLeo et al Pain 1994; 56: 9-16; Courteix et al Pain 1994; 57: 153-60; Aley et al; Slart et al Pain 1997; 69: 119-25; See Hargreaves et al Pain 1988; 32: 77-88 et al.

In vivo model of transient focal ischemia

In one aspect of the invention, (i) elevated potency of a compound at physiological pH to “disease-induced low pH” in cells expressing human NMDA receptors (eg, IC ₅₀ / “disease-induced low pH” at physiological pH). At IC ₅₀ ), repeating at least five efficacy-elevation experiments until the 95% confidence interval has not changed more than 15% by the addition of a new experiment, (ii) compounding in an animal model of transient local ischemia Testing and measuring the effect of the compound on infarct volume by repeating at least 12 experiments until the 95% confidence interval does not change by at least 5% with the addition of a new experiment; (iii) at least 5 in step (i) A method of identifying a chemical compound useful for treating ischemic injury in humans is provided by selecting a compound in which an increase in efficacy occurs and in step (ii) the compound decreases infarct volume by at least 30%. Thus, the candidate drug must meet or exceed ex vivo and in vivo criteria to be a good drug for human use, hi one embodiment, the cell comprises a NR1 subunit and at least one NR2 subunit of the NMDA receptor. In a further embodiment, the NR2 subunit may be an NR2B subunit In another embodiment, the NR2 subunit may be an NR2A subunit.

In another, more general aspect of the invention, (i) an increase in efficacy of at least 5 is shown in the experiment, wherein the increase in efficacy of the compound is a 95% confidence interval for increasing the efficacy of the compound at "disease induced pH" and physiological pH. The addition of this new experiment was repeated in the cell at least five efficacy boost experiments until no more than 15% unchanged, tested in cells, (ii) infarct volume measured in animal models of focal ischemia, with 95% confidence intervals At least 12 experiments were repeated until no more than 5% of the experiment was changed, and a compound was selected that treats a disease that lowers pH by activating a human NMDA receptor antagonist as measured and exhibiting a minimum 30% reduction. The method is provided. In one embodiment, the cell may express an NR1 subunit and at least one NR2 subunit of an NMDA receptor. In further embodiments, the NR2 subunit may be an NR2B subunit. In other embodiments, the NR2 subunit can be an NR2A subunit.

In a preferred embodiment, for infarct volume measured in an animal model of focal ischemia, at least 15 experiments are repeated until a 95% confidence interval has not changed by at least 10% by a new experiment, indicating a minimum 30% reduction. A method of selecting a compound or compound selected from one is provided. In another specific embodiment, “disease-induced low pH” may be associated with an ischemic disease such as stroke. In another embodiment, the middle cerebral artery occlusion model is an animal model of transient local ischemia in rodents such as mice. Can be used.

Local ischemic stroke can damage the brain by blocking the blood supply to each area of the brain. Local ischemic stroke is generally any one or more “primary cerebral arteries” (eg, the middle cerebral artery, anterior cerebral artery, posterior cerebral artery, internal carotid artery, spine), as opposed to a secondary artery or arteriole. Artery or basal artery). Thus, local ischemic stroke, as defined herein, is a model of cerebral embolism stroke (eg Bowes et al., Neurology 45: 815-819) in which a plurality of coagulation particles occlude secondary or small arteries. 1995).

Focal ischemia can be induced in any mammal, including, but not limited to, rodents, mice, rats, rabbits, gerbils, and the like (also Renolleau S, Stroke. 1998 Jul; 29 (7). ): 1454-60; see Gotti, B. et al., Brain Res, 1990, 522, 290-307). Gerbil, for example, is an experimental model that is widely used in the study of ischemic stroke since brain blood supply is controlled by only two common carotid arteries. Because they have an incomplete circle of Willis, they have special properties in Gerbil (Chandler et al., J. Pharmacol. Methods 14: 137-146, 1985; Finkelstein et al., Restor. Neurol. Neurosci. 1: 387-394, 1990; Levine and Sohn, Arch. Pathol. 87: 315-317, 1969; Kahn, Neurology 22: 510-515, 1972).

The test compound may be administered to the animal either before or after occlusion of the artery. In one embodiment, the test compound may be administered intraperitoneally. In one embodiment, the test compound may be administered intraventricularly. The test compound may be administered before occlusion of the artery, eg, about 10, 20, 30, 40, 50 or 60 minutes before ischemia. Alternatively, the test compound may be administered at about 10, 20, 30, 40, 50, 60, 90, or 120 minutes after occlusion of the artery, eg, ischemic phenomenon (ie, post-reperfusion), or It may be administered about 4, 6, 8, or 10 hours, or after about 1, 2, 3, 4, 5, 6, 7 or 8 days.

Evidence that the compound can protect cells in the ischemic region can be tested in animal models that experimentally occlude the middle cerebral artery (MCA), namely the middle cerebral artery occlusion model (MCAO). Such animal models for stimulating in vivo ischemia that may occur in human subjects are published in the industry. Experimental occlusion of MCA typically results in basal ganglion and huge unilateral ischemic regions associated with the frontal, parietal and temporal cortical regions (Menzies et al., Neurosurgery 31, 100-106 (1992)). Ischemic lesions grow over time, starting with smaller cores, at sites perfused by MCA. This penumbra, enclosed by the core infarction, is the tissue perfused from the core to the collateral circulation during occlusion, due to the propagation of the lesion. The effect of the therapeutic agent on the semishaded surrounded by the core of the ischemic phenomenon can be investigated by obtaining a brain slice from an animal. MCA supplies blood to the basal ganglia and inner capsules, as well as to the cortical surfaces of the frontal, parietal, and temporal lobes. Brain slices can be taken over the area where the greatest ischemic effect occurs. MCAO can be derived from any mammal, such as, but not limited to, mice, rats, rabbits and gerbils (also Renolleau S, Stroke. 1998 Jul; 29 (7): 1454-60; Gotti, B et al., Brain Res, 1990, 522, 290-307). The MCA model allows for indirect measurement of neuronal cell death following ischemia (ie occlusion of the left middle cerebral artery). In one embodiment, transient local cerebral ischemia of the middle cerebral artery can be used to test the compound.

Transient focal cerebral ischemia can be caused by intraluminal mid cerebral artery (MCA) occlusion. Occlusion may be by any means of blocking arteries, such as sutures such as monofilament sutures. After anesthesia, the probe can be anchored to the skull to monitor the relative changes in local cerebral blood flow. These changes can be monitored with a laser Doppler flow meter (Perimed). For example, in mice, probes can be anchored to the 2 mm posterior and 4-6 mm sides of bregma. The incision can then be made to access the MCA and the material can be inserted to occlude the MCA. For example, sutures may be performed to the internal carotid artery via an external carotid artery stump until the monitored blood flow stops. After an MCA occlusion cycle such as 30 minutes, 45 minutes or 60 minutes, the blocking material can be withdrawn to restore blood flow.

In another embodiment, a bilateral carotid occlusion model can be used to demonstrate that the compound can protect cells in the ischemic region. Animals may be anaesthetized, incisions may be made in the ventral neck to separate conventional carotid arteries, and complete occlusion for a period of time, for example, 5, 10, 15, 20, 30, 45 or 60 minutes. have. The artery may be occluded by any means, such as a clip such as a microaneurysm clip. The occlusion can then be stopped and the incision closed. In certain embodiments, bilateral carotid artery occlusion can be performed in gerbils.

After surgical manipulation, the animals can be restored. After the animal has survived for a period of time, for example about 12, 24, 36, 48 or 72 hours, the animal can be sacrificed and the brain removed, for example about 1, 2, 3, 4, It can be divided into 5 or 10 mm sections. The infarct volume can then be identified by staining the brain sections at 37 ° C. for about 20 minutes with a suitable dye such as 2% 2,3,5-triphenyltetrazolium chloride (TTC) in PBS. The infarct area of each section can then be measured and multiplied by the section thickness to obtain the infarct volume of the section. In addition, the ratio of contralateral to ipsilateral hemisphere section volumes can be multiplied by the corresponding infarct section volume for edema inhibition. Infarct volume can be determined by summing the frequency section thickness of the infarct area for all sections.

After testing the compound in an animal model of transient local ischemia and measuring the effect of the compound on infarct volume, a compound can be selected that reduces the infarct volume by at least 30%. In further embodiments, the infarct volume is at least 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, Compounds that reduce 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 65, 70, 75, 80, 85, 90, 95, 97, 99 or 100% can be selected. In a further embodiment, the compound is at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, at ischemic and physiological pH. 22, 23, 24, 25, 30, 40, or 50 shows an increase in potency (ie, phys pH / Isc pH), and as shown in FIG. 1, infarct volume is reduced to at least 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 65, 70, 75, 80, 85, 90, 95, 97, 99 or 100%, which numbers independently include any combination of these numbers, each combination deemed particularly disclosed. In certain embodiments of the invention, the mean, i.e., the sum of all observations divided by the number of observations, can be calculated for efficacy gain and infarct volume experiments, and the mean value of the compound is at least 5, 6 at ischemic and physiological pH. , 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23 may exhibit an elevated efficacy (ie, phys pH / Isc pH) , As shown in FIG. 1, the infarct volume is at least 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 , 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 65, 70, 75, 80 or 80%.

Infarct volume experiments can be repeated until the 95% confidence interval has not changed more than 10% with the addition of a new experiment. In another embodiment, the infarct volume experiment has a 95% confidence interval of about 25%, 24%, 23%, 22%, 21%, 20%, 19%, 18%, 17%, 16 with the addition of a new experiment. Repeat until no more than%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, or 2% can do. In a further embodiment, infarct volume experiments have 96%, 97%, 98% or 99% confidence intervals of about 25%, 24%, 23%, 22%, 21%, 20%, 19% with the addition of a new experiment. , 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3% or 2 Repeat until no more than% changes.

Other animal models of transient focal ischemia include, but are not limited to, intraarterial injection of microspare or coagulated blood in rats, occlusion of 4 vessels, occlusion of 2 vessels in gerbils, or clot formation photochemically induced by dissolution. It is not limited. Such models are known to those skilled in the art.

compound

In one aspect of the invention, the compounds identified by the methods provided herein are at least 10 times more selective in binding to human NMDA receptors than other glutamate receptors, other receptors disclosed herein. In a further or alternative embodiment, the compound has a therapeutic index of at least 2: 1 or greater.

In another embodiment, the compound is at least 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40 in binding to human NMDA receptors than other glutamate receptors. , 45, 50, 55, 60, 65, 70, 75, 78, 85, 90, 95, 100, 125, 150, 175, 200, 300, 400, 500, or 1000 times more selective, and the glutamate receptor The following glutamate receptors: AMPA GIuRl (GenEMBL accession number. X57497, Xl 7184, 157354), AMPA GluR2 (GenEMBL accession number. X57498, M85035, A46056), AMPA GluR3 (GenEMBL accession number. M85036, X82068), AMPA GluR4 (GenEMBL accession number.M36421, U16129), Kainate GluR5 (GenEMBL accession number.X66118, M83560, U16125), Kainate GluR6 (GenEMBL accession number.D10054, Zl 1715, U16126), Kainate GluR7 (GenEMBL accession number M83552, U16127), Kainate KA-I (GenEMBL accession number.X59996, S67803α), Kainate KA-2 (GenEMBL accession number.DlOOl 1, Zl 1581, S40369), Orphan dl GRIDl (GenEMBL accession) V. DlO 171, Z 17238), Orphan d2 GRID2 (GenEMBL Accession No. D13266, Z17239), and / or Group 1 GluRs, including metabotropic glutamate receptors (mGluRs), such as mGluR 1 and mGluR 5, , Group 2 mGluRs comprising mGluR 2 and mGluR 3, and Group 3 mGluRs comprising mGluR 4, mGluR 6, mGluR 7, and mGluR 8, including but not limited to. The NMDA receptor is any subunit thereof, for example, but not limited to, NMDA NRl (chromosome (human) 9q34.3, GenEMBL accession No. mouse: D10028, GenEMBL accession No. rat: X63255, GenEMBL language Session No. human: X58633), NMDA NR2A (chromosome (human): 16pl3.2, GenEMBL accession No. mouse: D 10217, GenEMBL accession No. rat: D13211, GenEMBL accession No. human: U09002); NMDA NR2B (chromosome (human): 12 pl2 GenEMBL accession No. mouse: D10651 'GenEMBL accession No. rat: M91562, GenEMBL accession No. human: U28861α); NMDA NR2C (chromosome (human) 17q24-q25, GenEMBL assay No. mouse: D10694, GenEMBL assay No. rat: D13212); NMDA NR2D (chromosome (human) 19ql3.1qter, GenEMBL accession No. mouse: D12822, GenEMBL accession No. rat: D13214, GenEMBL accession No. human: U77783); NMDA NR3A (GenEMBL Assay No. Rat: L34938 and / or NMDA NR3B, etc.) Alternatively, the compound may bind to human NMDA receptors at least 2, 3, or more than the other glutamate receptors disclosed above. 4, 5, 6, 7, 8, or 9 times more selective.

Additionally or alternatively, the compound may be at least 10 times more selective in binding to NMDA receptors than other receptor types. In another embodiment, the compound is at least 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40 in binding to the NMDA receptor than the other receptor types , 45, 50, 55, 60, 65, 70, 75, 78, 85, 90, 95, 100, 125, 150, 175, 200, 300, 400, 500, or 1000 times more selective, the other Receptor types include: dopamine receptors such as D1, D2, D3, D4 and D5; mu opioid receptors, such as opioid receptors such as mu1 and mu2; Delta opioid receptors such as delta 1 and delta 2 and kappa opioid receptors such as kappa 1 and kappa 2; Collinic receptors including muscarinic and nicotine receptors; Included include but are not limited to epinephrine receptors and epinephrine receptors, GABA receptors, such as, but not limited to, GABA-A and GABA-B receptors, such as those for the peptides listed in Table 2 below. It is not limited. Alternatively, the compound is not more selective for human NMDA receptors or at least 2, 3, 4, 5, 6, 7, 8 or 9 times more selective than the receptors described above.

In another embodiment, the compound binds to human NMDA receptors rather than serotonin receptors, at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40 , 45, 50, 55, 60, 65, 70, 75, 78, 85, 90, 95, 100, 125, 150, 175, 200, 300, 400, 500, or 1000 times more selective. Alternatively, the compound is not more selective or at least 2, 3, 4, 5, 6, 7, 8, or 9 times more selective in binding to human NMDA receptors than serotonin receptors. Serotonin receptors _{5HT 1A, 5HT 1B, 5HT 1D} , 5HT 1E, and 5HT ₁ comprising a 5HT _1F; 5HT ₂ comprising a 5HT _2A, 5HT _2B, and 5HT _2C; 5HT ₃ ; 5HT ₄ ; 5HT ₅ comprising a 5HT _5a and 5HT _5B; 5HT ₆ and 5HT ₇ , including but not limited to. In another embodiment, the compound is at least 10, 11, 12, 13, 14, 15, 16, 17, in binding to a human NMDA receptor than a histamine receptor comprising H1, H2, H3 and H4 histamine receptors. 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 78, 85, 90, 95, 100, 125, 150, 175, 200, 300, 400, 500, or 1000 times more selective. Alternatively, the compound is not more selective or at least 2, 3, 4, 5, 6, 7, 8, or more selective in binding to human NMDA receptors than histamine receptors including H1, H2, H3 and H4 histamine receptors. 9 times more selective. In another embodiment, the compound binds to human NMDA receptors rather than calcium channels, at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40 , 45, 50, 55, 60, 65, 70, 75, 78, 85, 90, 95, 100, 125, 150, 175, 200, 300, 400, 500, or 1000 times more selective.

Screening the compound to determine the affinity of the drug for a particular receptor is one of the important things in the drug discovery process. The method of determining receptor selectivity can be performed by any method known to those skilled in the art. Shielding can be used as a primary shielding method for large compound large compound libraries or as a secondary shielding to rank binding affinity for various receptor types or subtypes. In one embodiment, this analysis can be performed in a high throughput system, for example a filter-plate shielding system such as a Millipore Multiscreen ^™ _HTS filter plate.

In one embodiment, radioligand binding assays can be used to determine receptor selectivity for a particular receptor. In one specific embodiment, a saturation binding assay can be used to determine the binding constant (K _d ) of a test compound for a particular receptor. Saturation binding assays can be performed according to methods known in the art. In general, saturation binding assays can be performed by obtaining membranes of cells expressing specific receptors. For example, cells such as CHO cells can be transfected to express human NMDA receptors such as NR1 / NR2A or NR1 / NR2B human NMDA receptors. Alternatively, cells can be used to endogenously express human NMDA receptors such as NR1 / NR2A or NR1 / NR2B human NMDA receptors. In one embodiment, whole cell binding assays can be performed. Alternatively, after lysing the cells, the membrane can be separated from the cells, for example using centrifugation to obtain the membrane portion of the lysate (e.g., laboratory method for isolation). of cell membranes, A. Hubbard and Z. Conn The Journal of Cell Biology (1975) and Rogers et al., 1991, J. Neuroscience: 2713-2724). The whole cell or cell membrane can then be incubated with a series of dilutions of the radiolabeled ligand, ie the test compound, eg, 3H-labeled ligand. After incubation for a period of time, for example at least 1, 2 or 3 hours, the membrane may be washed several times, for example 5, 10, 15 or 20 times. Thereafter, scintillation fluid may be added and radioactivation of the cells or cells or membranes may be performed. Nonspecific binding can also be determined by separation experiments with excess unlabeled competitor ligand. Specific binding can be calculated by subtracting nonspecific activity from total activity. The binding constant (K _d ) can then be determined by nonlinear regression and Scatchard analysis, for example by adjusting specific binding by free ligand concentration, using Prism Data Software (www.Graphpad.com). . In addition, the number of binding sites (maximum binding dose (B _max )) can also be calculated using non-linear regression and Scatchard analysis, eg, prism data software.

In another embodiment, a substituted radioligand binding assay can be performed to determine the relative affinity value (IC ₅₀ ). Whole cells or isolated cell membranes expressing specific receptors can be used as described above. A series of dilutions of unlabeled competitor ligands can be used as a comparison to constant radioligand concentrations and control binding experiments to determine the inhibition without unlabeled ligand (% control). The relative affinity value IC ₅₀ can be determined by adjusting the binding inhibition value using nonlinear regression, for example prism data software.

The following compounds can be selected for conducting good human clinical trials in the treatment of disease lowering the area of infected tissue. Other compounds may be selected that satisfy the new parameters in accordance with the following guidance generally disclosed herein.

In one embodiment, the compound selected according to the processes and methods disclosed herein is selected from the group consisting of the following compounds and pharmaceutically acceptable salts, enantiomers, enantiomer mixtures thereof, and mixtures thereof:

및

And

.

.

Stereochemistry

The three-dimensional arrangement of the compounds is believed to play an important role in the activity and / or suitability of the compounds for therapeutic use. It has been experimentally observed herein that the enantiomers of the compounds may both be selected, or one is selected and one is not selected using the criteria described herein. Presumably, at a particular location, both enantiomers can be selected using the criteria provided.

In another embodiment, the compound selected according to the processes and methods described herein is an NMDA receptor channel blocker, including but not limited to, FR 115427, NPS 1506, penciclidine (PCP), remasemide, TCP, Or EAA-090. In another embodiment the compound selected according to the processes and methods described herein is an NMDA receptor glutamate site antagonist, such as, but not limited to, CGP 40116, D-CPPene, GPI3000 (NPC 17742), MDL 100,453, or Cellpotel (CGS 19755) In another embodiment, the compound selected according to the processes and methods described herein is an NMDA receptor glycine site antagonist, such as, but not limited to, 7-Cl-kinneurenate, HA966, MRZ 2/576, ZD9379, Gabe Stinel (GVl 50526), and lycostinel (ACEA 1021, 5-nitro-6,7-dichloro-l, 4-dihydro-2,3-quinoxalindioone).

In another embodiment, the compounds selected according to the processes and methods described herein are not disclosed in PCT Publication WO 02/072542.

Side Effect

In a further aspect of the methods and processes described herein, the compound does not exhibit substantial toxic side effects. Using human NMDA receptors over other non-human species, in vivo, particularly in human patient populations, can be used to effectively assess potency and potency enhancement of compounds using in vitro assays that minimize toxic side effects before compounds enter the in vivo setting. Can be.

Toxic side effects include excitement, hallucinations, confusion, discomfort, paranoia, delusions, psychotic syndrome, rotarod damage, amphetamine-like stereotype behavior, homology, psychotic memory impairment, impaired exercise, anti-anxiety effect, elevated blood pressure , Lowering blood pressure, rising pulse, lowering pulse, hematologic abnormalities, electrocardiogram (ECG) abnormalities, cardiac toxicity, heart palpitations, motor stimulation, neuromotor function, mood changes, short-term memory loss, long-term memory loss, arousal, sedation, pyramid Extrapyramidal side effects, ventricular tachycardia, prolonged repolarization, ataxia, cognitive impairment and / or ataxia-like syndrome.

In addition, in other embodiments, the compounds selected or identified according to the methods and procedures described herein have no substantial side effects associated with other classes of NMDA receptor antagonists. In one embodiment, these compounds have side effects associated with NMDA antagonists of glutamate sites, such as Cellpotel, D-CPPene (SDZ EAA 494) and AR-Rl 5896AR (ARL 15896AR), such as excitement, hallucinations, confusion and Inability to die (Davis et al. (2000), Stroke 31 (2): 347-354; Diener et al. (2002), J Neurol 249 (5): 561-568); Paranoia and delusions (Grotta et al. (1995), J Intern Med 237: 89-94); Psychotic similar syndrome (Loscher et al. (1998), Neurosci Lett 240 (l): 33-36); Low treatment rate (Dawson et al. (2001), Brain Res 892 (2): 344-350); Amphetamine-like stereotype behaviors (Potschka et al. (1999), Eur J Pharmacol 374 (2): 175-187) and the like are substantially not shown. In another embodiment, these compounds have side effects associated with NMDA antagonists at glycine sites, such as HA-966, L-701,324, d-cycloserine, CGP-40116, and ACEA 1021, such as marked memory impairment and motor function. No damage (Wlaz, P (1998), Brain Res Bull 46 (6): 535-540), and the like. Furthermore, in a further embodiment, these compounds have side effects associated with NMDA receptor channel blockers such as MK-801 and ketamine, such as pseudopsychiatric disorders (Hoffman, DC (1992), J Neural Transm Gen Sect 89: 1-10 ); Cognitive impairment (free play, cognitive memory and attention loss; Malhotra et al (1996), Neuropsychopharmacology 14: 301-307); Sizoprenia-like syndrome (Krystal et al (1994), Arch Gen Psychiatry 51: 199-214; Lahti et al. (2001), Neuropsychopharmacology 25: 455-467), and hyperactivity and stereotype elevation (Ford et al (1989) Physiology and behavior 46: 755-758).

In another or alternative embodiment, at least 2: 1, at least 3: 1, at least 4: 1, at least 5: 1, at least 6: 1, at least 7: 1, at least 8: 1, at least 9: 1, Minimum 10: 1, Minimum 15: 1, Minimum 20: 1, Minimum 25: 1, Minimum 30: 1, Minimum 40: 1, Minimum 50: 1, Minimum 75: 1, Minimum 100: 1 or Minimum 1000: 1 Have a treatment rate greater than or equal to Treatment rate is defined as the ratio of the dose required to produce a toxic or lethal effect to the dose required to produce a harmless or therapeutic response. This may be the relationship between a half effective dose (dose when 50% of the group is in effect on the drug in certain ways) and a half toxic dose (dose when 50% of the group is adversely affecting the drug). The higher the treatment rate, the safer the drug is considered. It simply indicates that a much higher dose is taken to induce a toxic response to produce a beneficial effect.

The adverse event profile of the compound can be determined by any method known to those skilled in the art. In one embodiment, motor impairment can be measured, for example, by measuring locomotor activity and / or rotorod performance. Rotorod experiments involve a measure of the persistence of which animals can stay on an accelerated rod. In another embodiment, memory impairment is, for example, a passive avoidance paradigm; Evaluation can be made using Sternberg memory scanning and paired words for short term memory, or delayed free regeneration of the phase for long term memory. In a further embodiment, the anti-anxiety-like effect can be measured, for example, in an elevated plus maze task. In another embodiment, cardiac function may be monitored by blood pressure and / or body temperature measurements, and / or electrocardiogram performance for side effects testing. In another embodiment, neuromotor function and arousal can be measured, for example, by analyzing the critical flicker fusion threshold, selection response time, and / or body sway. In another embodiment, the mood can be assessed using, for example, self-rating. In a further embodiment, the schizophrenic symptom can be assessed using, for example, PANSS, BPRS, and CGI, and side effects can be assessed on the HAS and S / A scales.

disease

In a further aspect of the invention there is provided a method of treating or preventing a disease causing or related to a pH difference in a tissue in a patient group by administering a compound selected according to the methods or procedures described herein. Any disease, condition or condition that causes a drop in pH can be treated according to the methods described herein.

Also provided is a method for alleviating the progression of an ischemic, hypoxic, or excitatory cascade associated with a pH drop by administering an effective amount of a compound exhibiting the properties described herein. Also provided is a method of reducing cell death associated with a drop in pH by administering a compound exhibiting the properties described herein. Also provided is a method of reducing behavioral deficits associated with ischemic phenomena associated with lowering pH by administering a compound exhibiting the properties described herein.

In one embodiment, there is provided a method of treating a group of patients with ischemic injury or hypoxia, or preventing or treating neurotoxicity associated with ischemic injury or hypoxia by administering a compound selected according to the methods or procedures described herein. do. In one particular embodiment, the ischemic injury may be a stroke. In another embodiment, the ischemic injury may be selected from, but is not limited to, traumatic brain injury, cognitive impairment after bypass surgery, cognitive impairment after carotid angiogenesis, and / or neonatal ischemia followed by hypothermic circulation arrest. .

In another specific embodiment, the ischemic injury may be vasospasm after subarachnoid hemorrhage. Subarachnoid hemorrhage is an abnormal condition in which blood accumulates below the membrane surrounding the brain and the arachnoid membrane. This area, called the subarachnoid space, usually contains cerebrospinal fluid. Accumulation of blood in the subarachnoid space and spasm of blood vessels resulting therefrom can cause strokes, seizures and other complications. The methods and compounds described herein can be used to treat a group of patients suffering from subarachnoid hemorrhage. In one embodiment, the methods and compounds described herein can be used to limit the toxic effects of subarachnoid hemorrhage, eg, toxic effects including stroke and / or ischemia that can result from subarachnoid hemorrhage. . In certain embodiments, the methods and compounds described herein can be used to treat a group of patients suffering from traumatic subarachnoid hemorrhage. In one embodiment, the traumatic subarachnoid hemorrhage may be due to head injury. In another embodiment, the patient group has natural subarachnoid hemorrhage.

In another embodiment, a method of treating a group of patients with neuropathic pain or related diseases is provided by administering a compound selected according to the methods or processes described herein. In certain embodiments, neuropathic pain or related diseases include peripheral diabetic neuropathy, postherpetic neuralgia, complex regional pain syndrome, peripheral neuropathy, chemotherapy-induced neuropathic pain, cancer neuropathic pain, nerves Pathological pain, HIV neuropathic pain, trigeminal neuralgia, and / or central post-stroke pain.

Neuropathic pain may be associated with signals that occur ectopically and occasionally in the absence of ongoing adverse events by pathological processes in the peripheral or central nervous system. Such dysfunction is allodynia, hyperalgesia, intermittent abnormal sensations, and spontaneous, burning, shooting, stabbing, paroxysmal or electrical sensations, which can be treated with conventional syndromes, such as the compounds and methods described herein. electrical-sensation, paresthesia, hyperalgesia and / or dysesthesia.

In addition, the compounds and methods described herein can be used to treat neuropathic pain resulting from pathological phenomena of the peripheral or central nervous system, including pathological ischemia; Infectious or toxic diseases from ongoing metabolism, infectious or endocrine diseases, such as diabetes mellitus, diabetic neuropathy, allodynia, amyloid polyneuropathy (primary and familial), neurons with monoclonal protein Disorders, vascular neuropathy, HIV epidemic, herpes zoster-shingle and / or post shingles neuralgia; Neuropathy associated with Guillain-Barre syndrome; Neuropathy associated with Fabry disease; Entrapment due to anatomical abnormalities; Tertiary and other CNS neuralgia; Malignancy; Inflammatory or autoimmune diseases, such as demyelinating inflammatory diseases, rheumatoid arthritis, systemic lupus erythematosus, Sjogren syndrome, and the like; And diseases of unknown cause, such as, but not limited to, idiopathic distal small-fiber neuropathy. Other causes of neuropathic pain that can be treated in accordance with the methods and compositions described herein include, exposure to, ingestion of toxins or drugs (such as arsenic, thallium, alcohol, vincristine, cisplatinum, and dideoxynucleosides). Or abnormalities of absorption, immunoglobulinemia (immuno-globulinemia), genetic abnormalities and amputations (including wire resection), but are not limited thereto. Neuropathic pain can also be caused from compression of nerve fibers, such as, for example, radiculopathy and carpal tunnel syndrome.

In another embodiment, a method of treating a group of patients with brain tumors is provided by administration of a compound selected according to the methods or processes described herein. In a further embodiment, there is provided a method of treating a group of patients with neurodegenerative disease by administering a compound selected according to the methods or procedures described herein. In one embodiment, the neurodegenerative disease may be Parkinson's disease. In another embodiment, the neurodegenerative disease may be Alzheimer's, Huntington's and / or Amyotrophic Lateral Scerosis.

In addition, compounds selected according to the methods or processes described herein can be used prophylactically to prevent or protect against diseases or neurological conditions as described herein. In one embodiment, patient groups with predisposition to ischemic symptoms, such as genetic predisposition, can be prophylactically treated with the methods and compounds described herein. In another embodiment, a group of patients exhibiting vasospasm can be prophylactically treated with the methods and compounds described herein. In a further embodiment, groups of patients undergoing cardiac bypass surgery can be prophylactically treated with the methods and compounds described herein.

In addition, methods are provided for treating the following diseases or neurological conditions by administration of a compound selected according to the methods or processes described herein. The disease or neurological condition; Chronic nerve injury, chronic pain syndrome, such as diabetic neuropathy, ischemia, ischemia due to transient or persistent vascular occlusion, seizures, suppression of clarity, restless leg syndrome, hypocarbonate, hypercarbonate, diabetic Ketoesidosis, fetal death, spinal cord injury, traumatic brain injury, overlapping epilepsy, epilepsy, hypoxia and the like; Perinatal hypoxia, (brain) concussion, migraine, hypocarbonate, hyperventilation, laccidosis, prenatal fetal birth, brain glycoma, and / or retinopathy, and the like.

Dosing / Formulation

Hosts, including mammals and particularly humans suffering from any of the diseases disclosed herein, may be formulated with an effective amount of a compound disclosed herein, or a pharmaceutically acceptable prodrug, ester, and / or salt thereof, optionally Can be treated by administration to a host in combination with an acceptable carrier or diluent. The active compound may be added to any suitable route, e.g., oral, parenteral, intravenous, intradermal, intramuscular, subcutaneous, sublingual, transdermal, bronchial, throat, nasal, cream or ointment, local, rectal, intraarticular It can be administered by intraoral, intramed, intravaginal, intraperitoneal, intraocular, inhalation, as a buccal or oral or nasal spray.

The compounds of the present invention can be used in the form of pharmaceutically acceptable salts derived from inorganic or organic acids. "Pharmaceutically acceptable salts" are within the scope of medical judgment and are suitable for use in contact with human and lower animal tissues without causing excessive toxicity, hypersensitivity, allergic reactions, and the like. It means suitable salts with a risk ratio. Pharmaceutically acceptable salts are well known in the art. For example, P. H. Stahl, et al., Describe pharmaceutically acceptable salts in detail in "Handbook of Pharmaceutical Salts: Properties, Selection, and Use" (Wiley VCH, Zurich, Switzerland: 2002). Salts can be prepared in situ during the final separation and purification of the compounds of the invention, or by separation by reacting the free base function with the appropriate organic acid. Representative acid addition salts include acetates, adipates, alginates, citrate, aspartates, benzoates, benzenesulfonates, bisulfates, butyrates, camphorates, camphor surfonates, digluconates, glycerophosphates, hemi Sulfate, heptanoate, hexanoate, fumarate, hydrochloride, hydrobromide, hydroiodide, 2-hydroxyethanesulfonate (isethionate), lactate, maleate, methanesulfonate, nicotinate , 2-naphthalenesulfonate, oxalate, pamoate, pectinate, persulfate, 3-phenylpropionate, picrate, pivalate, propionate, succinate, tartrate, thiocyanate, phosphate, glutamate, Bicarbonate, p-toluenesulfonate and undecanoate, and the like. . In addition, basic nitrogen-containing groups include methyl, ethyl, propyl, and lower alkyl halides such as butyl chloride, bromide and iodide; Dialkyl sulfates such as dimethyl, diethyl, dibutyl and diamyl sulfates; Long chain halides such as decyl, lauryl, myristyl and stearyl chloride, bromide, and iodide; Arylalkyl halides such as benzyl and phenethyl bromide and the like; You can quaternize using agents such as Water or oil-soluble or dispersible products can be obtained thereby. Examples of acids that can be used to form pharmaceutically acceptable acid addition salts include inorganic acids such as hydrochloric acid, hydrogen bromide, sulfuric acid and phosphoric acid, and organic acids such as oxalic acid, maleic acid, succinic acid and citric acid, and the like.

Base addition salts may be prepared by reacting a carboxylic acid-containing moiety with a suitable base such as a hydroxide, carbonate or bicarbonate of a pharmaceutically acceptable metal cation, or with ammonia, or an organic primary, secondary or tertiary amine, It can be prepared as is during the final separation and purification of the compounds of the present invention. Pharmaceutically acceptable salts include cations of alkali or alkaline earth metals such as lithium, sodium, potassium, calcium, magnesium and aluminum salts, and ammonium, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, Non-toxic quaternary ammonia and amine cations including, but not limited to, triethylamine, diethylamine, ethylamine, and the like. Other representative organic amines useful for forming base addition salts include ethylenediamine, ethanolamine, diethanolamine, piperidine, piperazine, and the like.

Pharmaceutically acceptable salts can also be obtained by standard reactions well known in the art, for example, by sufficiently reacting a base compound such as an amine with an appropriate acid yielding a physiologically acceptable anion. Alkali metal (eg sodium, potassium or lithium) or alkaline earth metal (eg calcium or magnesium) salts of carboxylic acids may also be prepared.

The formulations may conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy. All methods include the step of bringing into association the compound or a pharmaceutically acceptable salt or solvate thereof with the carrier which constitutes one or more accessory compounds. In general, formulations may be prepared by uniformly and intimately binding the active compound to a liquid carrier or a finely divided solid carrier or both, and then, if necessary, to form the product into the desired formulation.

The compound or pharmaceutically acceptable ester, salt, sorbate or prodrug may be mixed with other active substances that do not impair the desired action, or with other materials that complement the desired action. Solutions or suspensions used for parenteral, intradermal, subcutaneous, or topical applications include, for example, the following components: water for injection, saline solution, fixed oil, polyethylene glycol, glycerin, propylene glycol or other synthetic solvents. Sterile diluents such as; Antibacterial agents such as benzyl alcohol or methyl parabens; Antioxidants such as ascorbic acid or sodium bisulfide; Chelating agents such as ethylenediaminetetraacetic acid; Buffers such as acetate, citrate or phosphate and tonicity modifiers such as sodium chloride or dextrose and the like. Parenteral preparations may be placed in ampoules, disposable syringes or vials for repeated administration, made of glass or plastic. Preferred carriers for intravenous administration are physiological saline or phosphate buffered saline (PBS).

Pharmaceutical compositions of the invention for parenteral injection include pharmaceutically acceptable sterile aqueous or non-aqueous solutions, dispersions, suspensions or emulsions and sterile powders for conversion into sterile injectable solutions or dispersions. Examples of suitable aqueous and non-aqueous carriers, diluents, solvents or vehicles include water, ethanol, polyols (propylene glycol, polyethylene glycol, glycerol, etc.), suitable mixtures thereof, vegetable oils (such as olive oil) and ethyl oleate. Injectable organic esters. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.

These compositions may also contain adjuvants, including preservatives, wetting agents, emulsifiers, and dispersants. In order to prevent the action of microorganisms, various antibacterial and antifungal agents can be used, for example, parabens, chlorobutanol, phenol, sorbic acid, and the like. It is also desirable to include isotonic agents, such as sugars, sodium chloride, and the like. In order to prolong the absorption of injectable pharmaceutical formulations, it is possible to use agents which delay absorption such as aluminum monostearate and gelatin.

In some cases, in order to prolong the effects of the drug, it is sometimes desirable to slow the absorption of the drug from subcutaneous or intramuscular injection. This can be done using a liquid suspension of crystalline or amorphous material that exhibits low solubility in water. The rate of absorption of the drug, in turn, depends on the rate of dissolution that may depend on crystal size and crystalline form. Alternatively, the drug may be dissolved or suspended in an oil vehicle to prolong the absorption of the parenterally administered drug formulation.

In addition to the active compounds, the suspensions may contain suspending agents such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxy, bentonite, agar-agar Gar, tragacanth, and mixtures thereof.

In addition to inert diluents, the formulation composition may also include adjuvant such as wetting agents, emulsifying and suspending agents, sweetening agents, flavoring agents, and perfuming agents.

The active compound may also be in micro or nano encapsulated form with one or more excipients as needed.

Injectable depot forms are made by forming microencapsulated matrixes of drugs in biodegradable polymers such as polyacted-polyglycolide. Depending on the ratio of drug to polymer and the nature of the particular polymer used, the rate of drug release can be controlled. Examples of other biodegradable polymers include poly (orthoesters) and poly (unhydrides). Depot injectable preparations are also prepared by incorporating the drug into liposomes or microemulsions that are compatible with living tissue.

Injectable preparations may be sterilized, for example, by the addition of a bactericide in the form of a sterile solid composition which can be dissolved or dispersed in sterile water or other sterile injectable medium immediately before use or by filtration through a bacterial-retaining filter. can do. Injectable preparations, for example, sterile injectable aqueous or oleaginous suspensions may be prepared according to known methods using suitable dispersing or wetting agents and suspending agents. Sterile injectable preparations may be sterile injectable solutions, suspensions or emulsions in non-toxic, parenterally acceptable dilutions or solvents, such as solutions in 1,3-butanediol. Among the acceptable vehicles and solvents, Ringer's solution, U.S.P. And isotonic sodium chloride solutions can be used. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose, any bland fixed oil can be employed including synthetic mono or diglycerides. In addition, fatty acids such as oleic acid are used in the preparation of injectables.

Formulations for parenteral (including subcutaneous, intradermal, intramuscular, intravenous and intra-articular) administration include antioxidants, buffers, bacteriostats and solutes that are intended to be agents of blood and isotonicity of the intended recipient. Aqueous and non-aqueous sterile injectable solutions, which may contain; And aqueous and non-aqueous sterile suspensions, which may include suspending agents and thickening agents. The formulations may be present in unit or multiple dose containers, such as sealed ampoules and vials, and stored in lyophilize just prior to use, requiring only the addition of a sterile carrier such as saline, distilled water for injection. Can be. Extemporaneous injection solutions and suspensions can be prepared from sterile powders, granules and tablets of the kind described above.

Another method of preparation of the present invention involves binding the compounds described herein to polymers that have enhanced aqueous solubility. Examples of suitable polymers include polyethylene glycol, poly- (d-glutamic acid), poly- (l-glutamic acid), poly- (l-glutamic acid), poly- (d-aspartic acid), poly- (l-aspartic acid). Tic acid), poly- (l-aspartic acid) and copolymers thereof. The molecular weight of the polyglutamic acid preferably has about 5,000 to about 100,000, more preferably about 20,000 to 80,000, most preferably about 30,000 to 60,000. The polymer is bound via ester bonding to one or more hydroxyl groups of the epothilones of the invention using essentially the protocol described in US Pat. No. 5,977,163, which is incorporated herein by reference. Preferred binding sites include hydroxyl group leaving of carbon 21 in the case of the 21-hydroxy-derivatives of the present invention. Other binding sites include, but are not limited to, leaving a hydroxyl group of carbon 3 and / or carbon 7.

In another method of preparation, the compounds of the present invention can be linked to monoclonal antibodies. This strategy allows the targeting of the compounds of the invention to specific targets. General protocols for the design and use of bound antibodies are described in "Monoclonal Antibody-Based Therapy of Cancer" (by Michael L. Grossbard, ed. (1998)).

The compound of the invention may be administered in any suitable route of administration, for example, oral, parenteral, intravenous, intradermal, intramuscular, subcutaneous, sublingual, transdermal, bronchial, throat, intranasal, cream or ointment, topically, rectally Intraarticular, intraocular, intramedullary, vaginal, intraperitoneal, intraocular, inhalation may be administered as a buccal or buccal or nasal spray. However, the route of administration may vary depending on the condition and severity of diabetic vascular disease or ocular inflammation. The exact amount of compound administered to the host or patient will be the task of the physician performing this. However, the dosage used will depend on many factors, including the age, sex of the patient, the exact disease to be treated, and its severity.

The amount of active compound that can be combined with the carrier material to produce a single dosage form will vary depending upon the subject treated and the particular mode of administration. For example, formulations for intravenous use may contain about 1 mg / mL to about 25 mg / mL, preferably about 5 mg / mL to 15 mg / mL, more preferably about 10 mg / mL of the compound of the present invention. It can include the amount of. For the compositions of the present invention, typical dosage ranges are from about 0.001 mg / kg per day to about 2500 mg / kg per day. Preferably, the dose ranges from about 0.1 mg / kg per day to about 1000 mg / kg per day. More preferably, the dose ranges from 1 mg / kg, 2 mg / kg, 5 mg / kg, 10 mg / kg, 15 mg / kg, 20 mg, kg, 25 mg / kg, 30 mg / kg, per day, 35 mg / kg, 40 mg / kg, 45 mg / kg, 50 mg / kg, 100 mg / kg, 200 mg / kg, 300 mg / kg, 400 mg / kg, 500 mg / kg and any given in this range Between about two values of about 0.1 mg / kg per day to about 500 mg / kg per day. Doses for humans generally range from about 0.005 mg to 100 g per day. Alternatively, the dosage range according to the invention is from about 0.01 μM to 100 μM, preferably from about 0.1 μM to about 100 μM, which is the blood serum level of the compounds of the invention. Suitable values of blood serum levels according to the present invention are about 0.01 μM, about 0.1 μM, about 0.5 μM, about 1 μM, about 5 μM, about 10 μM, about 15 μM, about 20 μM, about 25 μM, about 30 μM, about 35 μM, about 40 μM, about 45 μM, about 50 μM, about 55 μM, about 60 μM, about 65 μM, about 70 μM, about 75 μM, about 80 μM, about 85 μM, about 90 μM, About 95 μM and about 100 μM, and any blood serum levels (eg, about 10 μM to about 60 μM) that fall within the range of any two values above.

Other forms of dose presentation provided in tablets or in separate units conveniently contain amounts of one or more compounds of the invention that may be effective in these dosage ranges, or between these ranges.

The compounds and formulations of the present invention may be selected from any standard formulation known in the art; It may be administered in the form of solid, semisolid, or liquid formulations, as well as subcategories of each of these forms.

Solid dosage forms for oral administration include capsules, caplets, tablets, pills, powders, lozenges and granules. In such solid dosage forms, the active compound comprises at least one inert, pharmaceutically acceptable excipient or carrier such as sodium citrate or dicalcium phosphate and / or a) starch, lactose, sucrose, glucose, mannitol, and salicylic acid. Such fillers or extenders; b) binders such as carboxymethylcellulose, alginate, gelatin, polyvinylpyrrolidinone, sucrose, and acacia; c) humectants, such as glycerol; d) disintegrants such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate; e) wetting agents, such as cetyl alcohol and glycerol monostearate; h) absorbents such as kaolin and bentonite clay; And i) lubricants such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof. In the case of capsules, tablets and pills, the formulation may also comprise a buffer.

Solid compositions of a similar type may also be used as fillers in soft and hard filled gelatin capsules, using excipients such as lactose or milk sugar and high molecular weight polyethylene glycols and the like.

Solid formulations of tablets, capsules, pills, and granules can be prepared with coatings and shells, such as enteric coatings and other coatings well known in the pharmaceutical preparation art. They may optionally contain a opacifying agent and may also be compositions that release only the active compound (s) or, preferably, in a delayed manner in the intestinal tract. Examples of embedding compositions that can be used include polymeric substances and waxes.

Tablets may be made by compression or molding, optionally with one or more accessory compounds. Compressed tablets may be prepared by compression of an active compound in free-flowing form, such as powders or granules, optionally with a binder, lubricant, inert diluent, lubricity, surface active or dispersant and compressed in a suitable machine. have. Molded tablets may be prepared by molding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent. Tablets may be optionally coated or scored and formulated to provide slow release or controlled release of the active compound therein.

Compositions for rectal or vaginal administration are preferably cocoa butter, polyethylene glycol or suppository waxes, which dissolve the compounds of the invention in a solid at ambient temperature but liquid at body temperature to dissolve in the rectal or vaginal cavity to release the active compounds. Suppositories that can be prepared by mixing with a suitable non-irritating excipient or carrier, such as.

Semi-liquid formulations include formulations that are structurally very soft and not solid, but highly viscous to be considered liquid. This includes creams, pastes, ointments, gels, lotions, and other semisolid emulsions containing the active compounds of the present invention.

Liquid formulations for oral administration include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs. In addition to the active compounds, liquid formulations may be prepared using inert diluents commonly used in the art, such as water or other solvents, solubilizers and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, Benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils (especially cottonseed, groundnut, corn, germ, olive, castor and sesame oil), glycerol, tetrahydrofurfuryl alcohol Fatty acid esters of polyethylene glycol and sorbitan, mixtures thereof, and the like.

Formulations containing a compound of the present invention may be administered through the skin in a device such as a transdermal patch. The patch may be made from a matrix such as polyacrylamide, polysiloxane, or with a semipermeable membrane made from a suitable polymer to control the rate at which the substance is absorbed into the skin. For other suitable transdermal patch formulations and configurations, see US Pat. Nos. 5,296,222 and 5,271,940, and also Satas, D., et al, "Handbook of Pressure Sensitive Adhesive Technology, 2nd Ed.", Van Nostrand Reinhold, 1989: Chapter 25, pp. 627-642.

Powders and sprays may contain, in addition to the compounds of the present invention, excipients such as lactose, talc, silicic acid, aluminum hydroxide, calcium silicate and polyamide powder, or mixtures of these materials. Sprays may additionally contain conventional propellants, such as chlorofluorohydrocarbons. Such excipients are described, for example, in "Handbook of Pharmaceutical Excipients, 3rd Ed.", A.H. Kibbe, Ed. (American Pharmaceutical Association and Pharmaceutical Press, Washington, DC, 2000), all of which are incorporated herein by reference.

In one embodiment, the active compounds of the present invention are prepared with a carrier to protect the compound against rapid removal or rapid release from the living body (eg, controlled release formulations including implants and microencapsulated delivery systems). do. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polyacrylic acid. Methods of preparing such formulations are apparent to those skilled in the art.

도 1은 도 2, 3 및 4를 합성화하여 나타내었다. 이는 테스트된 24 화합물, 20 화합물(83%)의 것이 본 발명의 범위를 벗어나고(어두운 영역으로 표시됨), 테스트된 화합물의 80% 이상이, 생체내 치료에 우수한 식별된 표준을 만족하지 못한다는 것을 나타낸다. 회색 음영 영역은 우수한 약물 작용에 대하여, 기준으로부터 식별된 바운드를 정의하는 영역을 나타낸다. 바운드의 범위에 속하는 약물들은 회색 차단 영역에서 평균값(에러바 아닌)을 갖는 것들이다. 화합물 93-4, 93-5, 93-41, 93-31의 평균은 NR1/NR2B에 대한 어두운 영역에 속한다. (-)MK801 및 케타민의 평균은 NR1/NR2A에 대한 어두운 영역에 속한다(도 4).
특히, 도 1에서, 일시적 국소 허혈 현상 후, 심볼로 표시된 화합물들에 대하여 상술한 바와 같이, C57Bl/6 마우스에서 경색 체적을 측정하였다. 약물을 뇌실내(ICV; 정방형) 또는 복강내 주입에 의해 상술한 바와 같이, 적용하였다. 에러바는 SEM이다. 경색 체적을, 짝지은 대조군에 비해 IP 투여에 대하여 대조 경색 체적의 퍼센트로서 직접 측정하였다. 대조군은 고체선으로 나타났다(단락선은 평균 대조 경색 +/- SEM을 나타냄). 오픈 심볼은, CNS 1102 (CN, 압티가넬 또는 Cerestat, Dawson et al., 2001), 덱스트로메토르판(DM, Steinberg et al., 1995), 덱스트로르판(DX; Steinberg et al., 1995), 레보메토르판(LM; Steinberg et al., 1995), (S)케타민(KT; Proescholdt et al., 2001), 메만틴(MM; Culmsee et al. 2004), 이펜프로딜(IF, Dawson et al. 2001), CP 101,606 (CP; Yang et al. 2003), AP7 (Swan 및 Meldrum, 1990), 셀포텔(CGS19755, Dawson et al., 2001), (R)HA966 (HA; Dawson et al., 2001), 레마세미드(RE, Dawson et al., 2001), 할로페리돌(O'Neill et al., 1998), 7-Cl-키뉴렌산(CK, Wood et al., 1992) MK801의 입체이성체(+MK 또는 -MK; Dravid et al., 제조)을 다양한 설치류 또는 토끼 허혈 모델에서 문헌에 설명된대로 투여함으로써, 경색 체적에 있어서의 감소를 나타낸다(하기 레퍼런스 참조). 경색의 퍼센트 감소는, 약물에 의한 신경 밀도의 퍼센트 감소를 측정하기 위해, 약물에서의 경색 체적 대 케타민 및 7-Cl-키뉴렌산을 제외한 모든 화합물에 대한 대조에 있어서의 경색 체적의 비로부터 계산하였다.
또한, 도 1에서, 화합물 93-4, 93-5, 93-8, 93-31, 93-40, 93-43, 93-97, (+)MK801, (-)MK801, 및 다른 모든 화합물에 대한 pH 6.9 대 7.6에서 효능 상승을, 상술한 바와 같이, 표 1 및 2에 보고된 실험의 수로 계산하였다. 이펜프로딜(IF), CP 101,606(CP)에 대한 pH 상승을 문헌으로부터 결정하였다(Mott et al., 1998).
실시예 5: 신경병성 통증의 생체내 모델에서의 평가
방법
동물군: 시술 당일 100 ± 10g 및 테스트 당일 250 ± 10g으로 무게를 단 수컷 스프라구-돌리 랫트(Hsd:Sprague-Dawley^®TMSD^®TM, Harlan, Indianapolis, Indiana, U.S.A.)를 케이지 당 3마리를 넣어두었다. 동물군은 먹이와 물에 자유롭게 드나들게 하였고, 12:12시간 낮/밤 스케쥴을 유지시켰다. 동물 집단을 21℃, 60% 습도로 유지하였다. 모든 실험은 통증 연구에 관한 국제협회(International Association for the Study of Pain)의 가이드라인에 따라 수행하였고, 미네소타 동물 보호 대학(University of Minnesota Animal Care) 및 미국 협회의 승인을 얻었다.
약물 및 용량 용액: 약물을 증류수 내 1% v/v DMSO 및 66% v/v PEG 400에 용해시켰다. 화합물을 i.p. 루트로 투여하였다.
만성 신경병성 통증의 유발: 척수 신경 결찰(SNL) 모델(Kim and Chung 1992 Pain 50:355-63.)을 사용하여 만성 신경병성 통증을 유발시켰다. 동물군을 이소플루란으로 마취시키고, 좌측 L5 트랜스버스(transverse) 과정을 제거하여, L5 및 L6 척수 신경을 6-0 실크 봉합으로 타이트하게 결찰시켰다. 그리고 나서, 상처(wound)를 내부 봉합 및 외부 스테이플로 봉합하였다. 상처 클립을 시술 10일 후에 제거하였다.
기계적 이질통 테스트: 무해성 기계적 감도에 대한 기준선 및 치료 후 값을, 업-다운법(up-down method)(haplan, Bach et al. 1994 J Neurosci Methods 53: 55-63)에 따라, 다양한 스티프니스(stiffness)(0.4, 0.7, 1.2, 2.0, 3.6, 5.5, 8.5, 및 15 g)로 8 셈메스-바인슈타인 필라멘트(8 Semmes-Weinstein filament) (Stoelting, Wood Dale, IL, USA)를 사용하여 평가하였다. 테스트 전에, 동물군을 천공 금속 플랫폼에 놓고, 최소 30분간 주변에 순응시켰다. 각 치료군에서, 각 동물에 대한 평균 및 평균에 대한 표준 오차(SEM)을 결정하였다. 이러한 자극은 일반적으로 고통스럽지 않다고 여겨지므로, 본 테스트에서, 반응성에 있어서의 충분한 손상 유도성 증가는 기계적 이질통의 척도로서 해석되었다.
실험 디자인: 약물 투여 전에, 본 프레이 기준선 측정을 각각 30분 및 24시간에서 하였다. 추가적인 본 프레이 측정을 30, 60, 120 및 240분에 수행하였다. 테스트에 대한 시각표(timeline)를 아래에 요약하였다. 실험군은: 비히클(증류수내 1% DMSO + 66% PEG 400, i.p., 4 ml/kg, n = 10)·30 mg/kg 화합물 93-31 테스트(i.p., 4 ml/kg, n = 10)·100 mg/kg 화합물 93-31 테스트(i.p., 4 ml/kg, n = 10)·30 mg/kg 화합물 93-97 테스트(i.p., 4 ml/kg, n = 10)·100 mg/kg 화합물 93-97 테스트(i.p., 4 ml/kg, n = 10)·100 mg/kg 가바펜틴(i.p., 4 ml/kg, n = 12)(총 랫트: 62)이었다.
실험 시각표

블라인딩 과정: 약물 용액을 행동 테스트를 수행하지 않는 다른 실험자에 의해 투여하였다.
데이터 분석: Prism™ 4.01(GraphPad, San Diego, CA, USA)을 사용하여, 통계적 분석을 행하였다. 손상된 포우의 기계적 이질통을, 비히클군내에서, 반대측 및 동측 포우에서 관찰된 값을 비교하여 결정하였다. 시간에 걸쳐 비히클군 손상된 포우 값의 안정성을, 순위에 의해 프리드맨 이원적 분산 분석(Friedman two-way analysis of variance)을 사용하여 테스트하였다. 각 시간 포인트에서 약물 효과를, 순위에 의해 크루스칼-왈리스 일원적 분산 분석(Kruskal-Wallis one-way analysis of variance)을 수행한 후, 던 포스트 호크 테스트(Dunn's post hoc test)에 의해 분석하였다.
결과
본 프레이 테스트
기계적 이질통의 테스트를 SNL 수술 14일 후, 개시하였다. 단일 약물 투여 후, 기준선(약물 투여 30분 전) 및 30, 60, 120 및 240분에, 손상(동측) 및 일반(반대측) 포우 모두에게서 테스트를 수행하였다.
기준선에서 모든 동물군은 손상된 포우에서 기계적 이질통을 나타내었다(표 2). 손상의 수준을 집단 사이에서 비교할 수 있었고, 손상된 포우의 본 프레이 역치 연구 전반에 걸쳐, 비히클 치료군의 일반 포우에서 관찰된 것과 상당히 달랐다(도 6). 도 6은, 비히클군에서의 동물은 연구 전체 기간 동안 눈에 띄는 기계적 이질통을 보였다는 것을 나타낸다. 도시된 것은 손상된 포우 및 비히클로 치료된 동물군의 일반 포우에서의 평균±SEM (n=10) 본 프레이 역치이다. 포우간의 차이는 모든 시간대에서 눈에 띄었다(Mann-Whitney 테스트). 화합물 93-31 및 93-97은 일반 포우에서 측정된 본 프레이 역치에 영향이 없었다(도 7 및 8). 도 7은 화합물 93-31이 일반 포우에서 본 프레이 역치를 변화시키지 않는다는 것을 나타낸다. 도시된 것은 비히클, 가바펜틴 또는 i.p. 투여된 30 및 100 mg/kg 용량의 화합물 93-31로 치료된 동물군의 일반 포우에서의 평균±SEM (n=10) 본 프레이 역치이다. 도 8은 화합물 93-97이 일반 포우에서 본 프레이 역치를 변화시키지 않는다는 것을 나타낸다. 도시된 것은 비히클, 가바펜틴 또는 i.p. 투여된 30 및 100 mg/kg 용량의 화합물 93-97로 치료된 동물군의 일반 포우에서의 평균±SEM (n=10) 본 프레이 역치이다.
표 2: - 손상된 포우 - 본 프레이 역치값

표 3: - 손상된 포우 - 통계적 분석 요약

화합물 93-31(100 mg/kg i.p.)로의 치료는, 투여 후 30분 및 60분에서 식별할 수 있는 진통(analgesia)을 발생시켰다(도 9). 도 9는 화합물 93-31(100 mg/kg)의 i.p. 투여가 기계적 이질통을 감소시킨다는 것은 나타낸다. 도시된 것은 비히클, 가바펜틴(참조 화합물) 또는 i.p. 투여된 30 및 100 mg/kg 용량의 화합물 93-31로 치료된 동물군의 손상된 포우에서의 평균±SEM (n=10-12) 본 프레이 역치이다. 포스트-호크 분석(Dunn 테스트)는 30분 및 60분에 화합물 93-31(100 mg/kg)과 비히클군 사이에 중요한 패어와이즈(pair-wise) 차이를 보였다(p<0.01). 60분, 120분 및 240분에서 가바펜틴의 효과 또한 두드러졌다(각각, p<0.001, p<0.01, p<0.01). 30 mg/kg의 화합물 93-31, 30 및 100 mg/kg의 화합물 93-97의 진통 효과는 연구한 어느 시점에서도 없었다. 본 연구에서 비히클군의 통계적 분석은, 기준선과 30, 60, 120 및 240분 시점 사이에 본 프레이 역치에 있어서, 눈에 띌만한 차이가 없었음을 나타내었다(Friedman two-way ANOVA).
추가적으로, i.p. 투여된 화합물 93-97은 SNL 랫트에서 기계적 이질통을 경감시키지 못했다. 도 10은 화합물 93-97(30 및 100 mg/kg)의 i.p. 투여가 본 프레이 역치에 영향이 없음을 보여준다. 도시된 것은 비히클, 가바펜틴(참조 화합물) 또는 i.p. 투여된 30 및 100 mg/kg 용량의 화합물 93-97로 치료된 동물군의 손상된 포우에서의 평균±SEM (n=10-12) 본 프레이 역치이다. 60분, 120분 및 240분에서 가바펜틴의 효과 또한 두드러졌다(각각, p<0.001, p<0.01, p<0.01).
본 연구에서 테스트된 동물군에서 어떠한 부작용이 관찰되었다(62 동물군 중 8). 관찰된 부작용은 라이딩(writhing) 및 스트레칭(stretching)이었다(8 관찰군). 이들 부작용은 i.p. 약물 투여 후, 처음 몇 분(~ 5분) 동안 가장 일반적으로 관찰되었다. 스트레칭/라이딩은, 비히클 i.p.로 치료된 동물군(3/10) 및 약물 용량에 의존성을 나타내지 않는 동물군을 포함하는 모든 연구군에서 관찰되었다. 이들 부작용의 심각성은 심하지 않았고, 연구로부터 동물군을 배제시키기에 충분한 종말점(endpoint) 측정을 방해하지 않았다. 표 1에 본 연구에서 관찰된 부작용을 요약하였다. 일부 부작용이 종말점을 측정하는 동안 관찰되었다. 가장 일반적인 것은, 일부 내장통(visceral pain) 또는 과민성의 신호일 수 있는 스트레칭/라이딩이었다. 이는 비히클 및 i.p. 약물 치료군에서 관찰되었다. 동물군의 서브셋에서 비히클의 i.p. 투여와 연관된 것으로 생각되었다. 이는 상대적으로 희박하고, 단명하였고(< 5분), 종말점의 측정을 방해할 만큼 규모가 크지 않았다.
표 1 : 부작용

화합물 93-31은 100 mg/kg에서 i.p. 투여되는 경우, 신경병성 통증의 SNL 모델에서 기계적 이질통을 경감하는 것으로 나타났다. 화합물 93-97은 본 연구에서 테스트된 용량에서(30 및 100 mg/kg) SNL 랫트의 기계적 이질통을 경감시킬 수 없었다. 화합물 93-31(100 mg/kg)은 참조 화합물 가바펜틴(100 mg/kg)보다 빠르게 작용을 개시하고(30분), 작용의 보다 짧은 지속성(60분)을 나타내었다. 100 mg/kg 용량의 화합물 93-31로 치료된 동물군에서 관찰된 피크 역치는 대략 일반 포우에서 관찰된 것의 절반이었다. 완전한 전도(reversal)를 보다 높은 용량의 화합물 93-31로 수행할 수 있다면, 이는 ED50 이 대략 100 mg/kg 임을 암시한다.
실시예 6 : 선택된 화합물의 pH 의존성
n-알킬 유도체 시리즈가 pH 의존성에 대해 테스트되었다.

실시예 7 : Xenopus oocyte 어세이에서 수용체를 함유하는 NMDA-NR2B의 길항작용에 대한 IC ₅₀ 을 결정함에 있어서 인간 대 랫트 수용체 cDNA
효능 상승을 비교하기 위하여 랫트 NMDA 및 인간 NMDA 수용체를 사용한 상술한 실시예의 신경병성 통증의 SNL 모델에 있어서, pH 6.9 및 7.6에서 몇몇 화합물들의 효능을, 생체외 스크리닝 및 테스팅법에 따라 평가하였다.길항작용에서 pH 의존성 효능 상승의 결정은, pH 6.9에서 수용체의 차단에 대한 IC₅₀을 pH 7.6에서 수용체의 차단에 대한 IC₅₀으로 나누는 것으로 정의하였다. 랫트 수용체로부터 얻은 pH 의존성 효능 상승은, 모든 화합물에 대하여 인간 수용체에 대해 얻어진 pH 의존성 효능 상승을 예견하지 않았다.
표 A : 랫트 대 인간 pH 효능 상승

상술한 발명은 명확성과 이해를 목적으로 상세히 설명되어 있으나, 본 명세서의 개시로부터, 당업자는 본 발명의 진정한 범위로부터 벗어나지 않고, 형태 및 상세한 것들의 다양한 변화를 줄 수 있을 것이다. FIG. 1 is a graph comparing the increase in vitro efficacy at pH 6.9 versus 7.6 for the selection of NMDA receptor antagonists and tissue infarct volume reduction in control treatment in C57B1 / 6 mice following transient or persistent local ischemic events. Drugs were applied intraventricularly (ICV; 1 microliter of 0.5 mM; square solids) or intraperitoneally injected (IP, circular solids; NP93-4, 30 mg / kg; NP93-5, 10-30 mg / kg; NP93-40, 10-30 mg / kg; NP93-8, 30 mg / kg; NP93-31, 3 mg / kg). Error bars are SEM. Infarct volumes in drug treated animal groups were measured directly and infarct volumes in vehicle injected control mice were expressed as percentages. Open symbols include CNSl 102 (CN, Aptiganel or Cerestat, Dawson et al., 2001), dextromethorphan (DM, Steinberg et al., 1995), dextrose (DX; Steinberg et. al., 1995), Levomethorphan (LM; Steinberg et al., 1995), (S) Ketamine (KT; Proescholdt et al., 2001), Memantine (MM; Culmsee et al. 2004), Ipenpro Dill (IF, Dawson et al. 2001), CP101,606 (CP; Yang et al. 2003), AP7 (Swan and Meldrum, 1990), Cellpotel (CGS19755, Dawson et al., 2001), (R) HA966 (HA; Dawson et al., 2001), remasemid (RE, Dawson et al., 2001), haloperidol (O'Neill et al., 1998), 7-Cl-kinyurenic acid (CK, Wood et al. , 1992) and the reduction of infarct volume by administration of stereoisomers of MK801 (+ MK or -MK; Dravid et al.). The percent reduction in infarct was calculated from the ratio of infarct volume for all compounds except ketamine to determine the percent reduction in neuronal density. The pH rise for iffenprodil and CP101,606 was determined from Mott et al., 1998. Efficacy elevations for inhibition of NR1 / NR2B containing NMDA receptors at pH 6.9 vs. 7.6 for all other compounds except competitive antagonists were calculated as described herein and evaluated in two experiments (see Table 3 below). Drugs belonging to shades of gray are excellent ones with potential for in vivo safety and efficiency.
FIG. 2 is a graph comparing the in vitro potency improvement of selected compounds at pH 6.9 versus 7.6 for tissue infarct volume protection when the test drug was applied intraventricularly (ICV; square solid). Gray shade is the area representing the identified boundaries of the criteria for good drug action.
FIG. 3 compares the in vitro potency improvement of selected compounds at pH 6.9 versus 7.6 for tissue infarct volume protection when the test drug was applied by intraperitoneal infusion (IP; round solid). Gray shade is the area representing the identified boundaries of the criteria for good drug action.
4 compares the in vitro potency improvement at pH 6.9 versus 7.6 for tissue infarct volumes of selected compounds. Gray shade is the area representing the identified boundaries of the criteria for good drug action. The right panel shows the comparison for NR1 / NR2A and the left panel shows the comparison for NR1 / NR2B.
FIG. 5 shows the effects of Compounds 93-31 and (+) MK-801 on locomotor activity in rats, quantified as light breaks counted with a computer during a 2 hour period after 1 hour of purification. . The locomotor activity index is the total number of ray shorts during the trial divided by 1000.
FIG. 6 shows damaged paws showing substantial allodynia in an animal model of neuropathic pain. Animals in the vehicle group exhibited notable mechanical allodynias throughout the study. Shown are the mean ± SEM (n = 10) von Frey thresholds in damaged and normal paws of vehicle treated animals. The difference between the pows is noticeable at all time points (Mann-Whitney test).
Figure 7 shows that Compound 93-31 (i.p. administration) had no effect on normal pow. Shown are vehicle, gabapentin or i.p. Mean ± SEM (n = 10-12) von Frey threshold in normal paws of 30 and 100 mg / kg doses of Compound 93-31 treated animals administered.
8 shows that compound 93-97 (i.p.) had no effect on normal pow. NeurOp 93-97 does not change the Bone Frey threshold on normal PoE. Shown are vehicle, gabapentin or i.p. Mean ± SEM (n = 10-12) von Frey threshold in normal paws of 30 and 100 mg / kg doses of Compound 93-97 treated animals administered.
9 shows i.p. Compound 93-31 (100 mg / kg) administered shows that alleviation of mechanical allodynia in rat spinal nerve ligation (SNL) model. Treatment with compound 93-31 (100 mg / kg i.p.) resulted in noticeable analgesia at 30 and 60 minutes after administration. At any time point studied, 30 mg / kg of compound 93-31 and 30 and 100 mg / kg of 93-97 had no analgesic effect. In this study, statistical analysis of the vehicle group showed no significant difference between baseline and 60, 120, and 240 minute time points (Friedman two-way ANOVA).
10 shows i.p. Compounds 93-31 (100 mg / kg) administered reduce the mechanical allodynia of SNL rats. Compound 93-31 I.P. of the test compound. Administration reduced mechanical allodynia. Shown are vehicle, gabapentin (reference compound) or i.p. Mean ± SEM (n = 10-12) von Frey threshold in injured paws of 30 and 100 mg / kg doses of Compound 93-31 treated animals administered. Post-hawk analysis (Dunn test) showed noticeable pair-wise differences between compound 93-31 (100 mg / kg) and vehicle group at 30 and 60 minutes. In addition, the effects of gabapentin were noticeable at 60, 120 and 240 minutes (p <0.001, p <0.01, and p <0.01, respectively).
FIG. 11 compares the in vitro potency increase at pH 6.9 versus 7.6 for fold increase in pain threshold in rodent spinal nerve ligation models. Efficacy boost was determined for each compound described herein. Pain thresholds were measured after administration of compounds 93-31. Pain threshold values are conventionally defined as IF (ifenprodil, De Vry et al, Eur J Pharmacol 491: 137-148, 2004), K (ketamine, Chaplan et al. JPET 280: 829-838 1997), CP (CPlOl, 606, Boyce et al. Neduropharmacol 38: 611-623, 1999), MK (MK801, Chaplan et al. JPET 280: 829-838 1997), D (dextrorphan, Chaplan et al. JPET 280: 829-838 1997), DM ( dextromethorphan, Chaplan et al. JPET 280: 829-838 1997), and M (memantine, Chaplan et al. JPET 280: 829-838 1997). Gray shade is the area representing the identified boundaries of the criteria for good drug action.
Example
The following examples are provided for illustrative purposes and do not limit the scope of the invention thereto.
Example
Example 1: Selectivity of Compound 93-4 for NMDA Receptor vs. Other Glutamate Receptors
Compound 93-4 series was injected into AMPA receptor and kainate receptor subunitsXenopus oocyteIt is shown to be selective for the NMDA receptor due to lack of effect on. Glutamate or domoate induced current recording was performed using 2 μM compound 93-4 administered with a 2-electrode voltage clamp, and an antagonist (glutamate for AMPA receptor, domoate for kinate receptor). No decrease in antagonist induced response was observed, indicating that compound 93-4 does not inhibit AMPA and catenate receptors. In addition, when the receptor was composed of NR1 / NR2B subunits and did not contain the NR1 / NR2A or NR1 / NR2D receptors, 3 μM of compound 93-4 was effective at inhibiting NMDA receptor mediated current.
Example 2: Effect of 93 Series Compounds on Motor Activity of Rats
In order to quantify motor activity with light shorts, 100-150 gm Sprague-Dawley rats were purified for 1 hour in an active box equipped with an optical monitor, followed by 93-4, 93-5, IP injections at 93-8, 93-31, 93-40, 93-41. After infusion, motor activity was monitored for 2 hours. Both stereoisomers of MK801 were used as positive controls. (+) MK801 showed an increase in initial motor activity followed by ataxia, indicating a normalized biphasic effect on motor activity. The data showed that the efficacy decreased at least 10-fold, in that (-) MK801 caused the induction of motor activity compared to the (+) MK801 compared to the vehicle injected control animals. In addition, 3-300 mg / kg 93-4, 3-300 mg / kg 93-5, 30-300 mg / kg 93-8, 3-300 mg / kg 93-31 (FIG. 5), 30 mg 93-40 / kg and 93-41 at 30-300 mg / kg had no noticeable effect on motor activity. The dose of 93 series compounds known to be neuroprotective did not affect motor activity.
Example 3: Xenopus oocyte Determination of pH Dependency Efficacy Shift in
Expression of NMDA receptors in Xenopus oocytes. 
cRNA was synthesized from linearized template cDNA for NMDA receptor subunits (NR1-1a, NR2B, NR2A) according to the manufacturer's specification (Ambion :). The cDNAs used correspond to GenBank numbers U08261 and U11418 (NR1-1a), AF001423 and CD13211 (NR2A), U11419 (NR2B). Briefly, cDNA was linearized to the appropriate restriction enzyme downstream of the code region, purified and incubated with RNA polymerase and appropriate concentrations of ribonucleotides. In vitro transcription cRNA was purified by standard methods. The quality of the synthesized cRNA was evaluated by gel electrophoresis, and the yield was evaluated by spectrophotometer and gel electrophoresis. Stage V and VI oocytes are removed from the large ovary, well cultured, and healthyXenopus laevisWas anesthetized with 3-amino-benzoic acid ethyl ester (1 gm / l). Clusters of oocytes were either 292 U / ml Worthington (Freehold, NJ) type IV collagen or 1.3 mg / ml collagen (Life Technologies, Gaithersburg, MD; 17018-029), 115 NaCl, 2.5 KCl, And Ca consisting of 10 HEPES, pH 7.5, in mM²⁺-Incubate for 2 hours in glass solution with gentle stirring to remove the follicular cell layer. The oocytes were then 1.8 mM CaCl₂Wash extensively in the same solution supplemented with 88 NaCl, 1 KCl, 24 NaHCO₃, 10 HEPES, 0.82 MgSO₄ , 0.33 Ca (NO₃)₂, And 0.91 CaCl₂ (In mM) and maintained in Barth's solution supplemented with 100 ug / ml gentamycin, 40 ug / ml streptomycin, and 50 ug / ml penicillin. Oocytes are manually defolliculate and injected into 5 ng NRl subunits and 10 ng NR2 subunits in a 50 nl volume within 24 hours of departure, and in bath solution, 18 ° C., 3-7 days. Incubated. The tip size of the glass injection pipette was 10-20 microns and the back was filled with mineral oil.
Preparation of pH-Dependent NMDA Receptor Antagonists for Testing. 
NMDA receptor antagonists are typically made into a 20 mM solution in 100% DMSO and stored at -20 ° C. This stock solution was all diluted in 2%, 0.2 mM, and 0.02 mM (1/10 v / v) sequentially in 100% DMSO. This mother liquor was then added to 90 mM NaCl, 3 mM KCl, 5 mM HEPES, 0.5 mM BaCl.₂Diluted in a suitable concentration range in a working solution consisting of, 10 uM EDTA, 100 uM glutamate, 50 uM glycine (pH 6.9 or 7.6 adjusted to NaOH or HCl, if necessary). The concentrations of the tested drug were 0.01, 0.03 micromoles (diluted 0.02 mM stock to an appropriate volume), 0.1, 0.3 micromoles (diluted 0.2 mM stock to an appropriate volume), 1, 3 micromoles (2 mM stock) Dilution to an appropriate volume), and / or 10, 30, 100 micromoles (diluted 20 mM mother liquor to an appropriate volume).
Voltage-clamp recordings from Xenopus oocytes. 
Two-electrode voltage clamp recordings were made 2-7 days after implantation. The oocytes were placed in a dual-track plexiglass recording chamber to spread the two oocytes, with a single perfusion line dividing in the Y configuration. . Dual recordings were performed using a 2-Warner OC725B two-electrode voltage clamp amplifier arranged at room temperature as recommended by the manufacturer. Glass micro electrodes (1-10 megohms) were filled with 300 mM KCl (voltage electrode) or 3 M KCl (current electrode). The bath clamp was connected through a silver chloride wire placed on each side of the recording chamber, assuming both were at a reference potential of 0 mV. Oocytes were harvested at 90 NaCl, 1 KCl, 10 HEPES, and 0.5 BaCl.₂(Unit: mM), sparged with a solution consisting of pH 7.3 and maintained at -40 mV. Final concentrations for the control application of glutamate (100 micromoles) and glycine (50 micromoles) were performed by adding appropriate volumes from 100 mM and 30 mM mother liquors, respectively. In addition, by adding a 1: 1000 dilution of 10 mM EDTA, 10 micromoles of the final EDTA was added to Zn.²⁺To complex the divalent ionic contaminants, such as were obtained. The external pH was adjusted to 6.9 or 7.6. Dose response curves were obtained by applying to successive fashion maximal glutamate and glycine, followed by adding varying concentrations of antagonists to glutamate / glycine. Dose response curves consisting of 4 to 6 concentrations were obtained in this manner. Baseline leak currents were measured at −40 mV before and after full recording, linearly corrected for any change in recording and leakage current. Oocytes with glutamate-induced responses less than 100 nA at pH 7.6 or 50 nA at pH 6.9 were not included. The level of inhibition by the applied antagonist is expressed as a percentage of the initial glutamate response and averaged across oocytes from a single frog. Each experiment included records at each pH of 3-10 oocytes from a single frog. . Calculate the average percent response at each of the 4 to 8 antagonist concentrations, (100-min) / (1 + ([conc] / IC₅₀)^nH) min, where min is the residual percent response in the saturated antagonist and IC₅₀Is the concentration of the antagonist causing half of the achievable inhibition and nH is the slope factor indicating the steepness of the inhibition curve. min can be defined as equal to or greater than zero. Min can be set to zero in an experiment with a known channel blocker. IC obtained at pH 7.6 and 6.9₅₀ Value is IC₅₀The ratios are shown as averaged together and to determine the average shift in.
Example 4 Determination of Neuroprotective Action in an In Vivo Model of Transient Focal Ischemia
Transient focal ischemia
Transient focal ischemia was caused by monofilament closure and intraluminal mid cerebral artery (MCA) occlusion. Briefly, male C57BL / 6 mice (The Jackson Laboratory, 3-5 months old) were anesthetized with 2% isoflurane in 98% O 2. The rectal temperature was controlled at 37 ° C. with a thermostatic blanket. Relative changes in local cerebral blood flow were monitored with a laser Doppler flow meter (Perimed). To do this, the probe was fixed directly to the skull, 2 mm posterior and 4-6 mm laterally of the bregma. 11-mm 5-0 Dermalon or Look (SP185) with tip flame-rounded black nylon nonabsorbable suture into the left internal carotid artery, until monitored blood flow stops (10.5-) 11 mm suture) through an external carotid artery stump. Thirty minutes after MCA occlusion, sutures were released to restore blood flow. After 24 h survival, the brain was removed and cut into 2 mm sections. Injuries were identified for 20 minutes at 37 ° C. in 2% 2,3,5-triphenyltetrazolium chloride (TTC) in PBS. The infarct area of each section was measured by NIH IMAGE (Scion Corporation, Beta 4.0.2 release) and multiplied by the section thickness to determine the infarct volume of each section. The density slice option in NIH IMAGE was used to segment the image based on intensity determined as 70% or 75% in the intact cortex on the opposite side. This criterion was maintained in all animal groups throughout the analysis and only emphasized the purpose at this intensity for area measurements. The area of injury is directly outlined, as identified by the reduction of the threshold electronically identified in the TTC dye. The ratio of contralateral to ipsilateral hemisphere section volumes was multiplied by the corresponding infarct section volume to correct edema. Infarct volume was determined by summing the infarct area number section thickness for all sections. At least 12 animals were included in each measurement. For some experiments, the area of damage was measured directly, at the discretion of the area of the reduced dye. The same result was obtained in both procedures.
Intraperitoneal administration of pH dependent NMDA receptor antagonists.
C57Bl / 6 mice received intraperitoneal (IP) administration of 93-4, 93-5, 93-8, 93-31, 93-40 30 minutes prior to MCA occlusion. A 30 mg / ml mother liquor in 50% DMSO was prepared by adding 30 mg of compound to 0.5 ml of DMSO followed by stirring with 0.5 ml of 0.9% saline.
The working solution for the IP injection solution was 3 mg / ml (50% v / v DMSO) in 0.9% saline, transferring 0.2 ml of mother liquor into a new tube, and drawing 0.9 ml of DMSO and 0.9 ml of 0.9% saline. Stirred and prepared. The final dose of 3-30 mg / kg was administered to the mice in an injection volume that varied depending on the weight of the animal and the desired dose.
Intraventricular administration of pH dependent NMDA receptor antagonists.
In a separate set of experiments, mice were injected intraventricularly (ICV) with NMDA antagonists (93-5, 93-97, 93-31, 93-41, 93-43) or a suitable vehicle prior to surgery. . Initially 20 mM mother liquor in 100% DMSO was prepared for all drugs. Five microliters of this mother liquor were transferred to a new tube and 45 microliters of DMSO was added with stirring for Drugs 93-41, 93-43. 150 microliters of phosphate buffered saline (PBS, 0.9% NaCl, pH 7.4, Sigma 1000-3) was then added to obtain a 0.5 mM drug solution in 25% (v / v) DMSO. For all other drugs, 5 microliters of 20 mM DMSO stock solution was transferred to a new tube and 15 ul of DMSO was added with agitation. 180 microliters of PBS was added to this solution to obtain a 0.5 mM working solution in 10% v / v DMSO. For vehicle, DMSO was replaced with 20 mM drug in DMSO. ICV injections were all performed in the right ventricle (2 mm posterior and 1 mm lateral, 3 mm needle insertion) of male C57BL / 6 mice (The Jackson Laboratory, 3-5 months of age) 30 minutes prior to MCA occlusion. . Mice were killed 24 hours after MCA occlusion, lesions were identified and analyzed as above. Subarachnoid hemorrhages were identified as blood clotting phenomena greater than ˜1 mm at the base of the skull and excluded.
result
Compounds 93-97, 93-43, 93-5, 93-41, and 93-31
2 shows a comparison of the ex vivo potency enhancement of these compounds at pH 6.9 versus 7.6 for tissue infarct volumes following ICV administration of compounds 93-97, 93-43, 93-5, 93-41, and 93-31. Indicated. The data shows the percent infarct volume determined for the vehicle injected control and the potency increase measured as described above. Gray shaded areas represent areas that define the bounds identified from the criteria for good drug action. Drugs that fall within the bounds are those that have an average (not error bar) in the gray blocking area.
For each compound the infarct volume in C57Bl / 6 mice following transient focal ischemia as described above was measured. Compounds 93-97, 93-43, 93-5, 93-41, and 93-31 were applied intraventricularly (ICV; solid circles) as described above. Error bars are the standard error of the mean (SEM). Efficacy elevations at pH 6.9 versus 7.6 for compounds 93-97, 93-43, 93-5, 93-41, and 93-31 were calculated as described for oocytes expressing NR1 / NR2B receptors.
Compound 93-4, 93-5, 93-8, 93-31, 93-40, (-) MK801 and (+) MK801
3 shows a comparison of the ex vivo potency elevation of compounds 93-4, 93-5, 93-8, 93-31, 93-40 at pH 6.9 versus 7.6 for tissue infarct volume. The data represent the actual infarct volume expressed in percent of that in the vehicle injected into the control animal group, and the increase in efficacy was calculated as described above. Gray shaded areas represent areas that define the bounds identified from the criteria for good drug action. Drugs that fall within the bounds are those that have an average (not error bar) in the gray blocking area.
For each compound the infarct volume in C57Bl / 6 mice following transient focal ischemia as described above was measured. The drug was applied by intraperitoneal injection as described above. Error bars are SEM. Infarct volume was inferred from the percent reduction in infarct volume for IP administration as compared to the paired control. This is the mean control infarct volume (mm) for the ICV experiments, represented by independent experiments and solid lines (paragraphs represent mean control infarct + -SEM).³Calculated as the product of infarct volume, expressed as a percentage of control infarction induced by the drug in. Oocytes expressing NR1 / NR2B receptors exhibiting potency enhancement at pH 6.9 vs. 7.6 for compounds 93-4, 93-5, 93-8, 93-31, 93-40, (-) MK801 and (+) MK801 Calculation as described in
Additional compounds
4 shows a comparison of the ex vivo potency elevation of NR1 / NR2A and NR1 / NR2B of known compounds at pH 6.9 vs. 7.6 versus percent control tissue infarct volume. Gray shaded areas represent areas that define the bounds identified from the criteria for good drug action. Drugs that fall within the bounds are those that have an average (not error bar) in the gray blocking area.
Open symbols include CNS 1102 (CN, Aptiganel or Cerestat, Dawson et al., 2001), dextromethorphan (DM, Steinberg et al., 1995), dextropan (DX; Steinberg et al., 1995), Levomethorphan (LM; Steinberg et al., 1995), (S) Ketamine (KT; Proescholdt et al., 2001), Memantine (MM; Culmsee et al. 2004), Iphen Prodil (IF, Dawson et al. 2001), CP 101,606 (CP; Yang et al. 2003), AP7 (Swan and Meldrum, 1990), Cellpotel (CGS19755, Dawson et al., 2001), (R) HA966 (HA; Dawson et al., 2001), remasemid (RE, Dawson et al., 2001), haloperidol (O'Neill et al., 1998), 7-Cl-kinyurenic acid (CK, Wood et al. , 1992) administration of the stereoisomer of MK801 (+ MK or -MK; Dravid et al., Manufactured) as described in the literature in various rodent or rabbit ischemia models, indicating a decrease in infarct volume (see reference below). . The percent reduction in infarction was calculated from the ratio of infarct volume in the drug to the control for all compounds except ketamine and 7-Cl-quinuric acid to determine the percent reduction in nerve density by the drug. .
Efficacy elevations at pH 6.9 vs. 7.6 for all compounds were calculated as described above in oocytes expressing NR1 / NR2A or NR1 / NR2B receptors (see Table 3 and Table 4 for a summary of the experimental numbers). The pH rise for iffenprodil (IF), CP 101,606 was determined from the literature (Mott et al., 1998).
The number of mice examined for infarct volume is shown in Table 3. In measuring potency elevation at the NR1-1a / NR2B receptor, the number of frogs used and the largest number of oocytes tested were shown in Table 3 at a single concentration of pH 6.9 and pH 7.6. In determining the IC 50 at each pH, various concentrations of each drug were tested. In measuring potency elevation at the NR1-1a / NR2A receptors, at a single concentration of pH 6.9 and pH 7.6, the number of frogs used and the largest number of oocytes tested are shown in Table 4.

FIG. 1 shows the synthesis of FIGS. 2, 3 and 4. This indicates that 24 compounds tested, 20 compounds (83%) are outside the scope of the present invention (indicated by the dark areas), and at least 80% of the tested compounds do not meet the identified standard of excellence for in vivo treatment. Indicates. Gray shaded areas represent areas that define the bounds identified from the criteria for good drug action. Drugs that fall within the bounds are those that have an average (not error bar) in the gray blocking area. The average of compounds 93-4, 93-5, 93-41, 93-31 belongs to the dark region for NR1 / NR2B. The mean of (-) MK801 and ketamine belongs to the dark region for NR1 / NR2A (FIG. 4).
In particular, in FIG. 1, infarct volume was measured in C57Bl / 6 mice, as described above for compounds represented by symbols after transient focal ischemia. The drug was applied as described above by intraventricular (ICV; square) or intraperitoneal injection. Error bars are SEM. Infarct volume was measured directly as a percentage of control infarct volume for IP administration compared to the paired control. Controls appeared as solid lines (short lines represent mean control infarct +/- SEM). Open symbols include CNS 1102 (CN, Aptiganel or Cerestat, Dawson et al., 2001), dextromethorphan (DM, Steinberg et al., 1995), dextropan (DX; Steinberg et al., 1995), levomethorphan (LM; Steinberg et al., 1995), (S) ketamine (KT; Proescholdt et al., 2001), memantine (MM; Culmsee et al. 2004), iffenprodil (IF , Dawson et al. 2001), CP 101,606 (CP; Yang et al. 2003), AP7 (Swan and Meldrum, 1990), Cellotel (CGS19755, Dawson et al., 2001), (R) HA966 (HA; Dawson et al., 2001), remasemid (RE, Dawson et al., 2001), haloperidol (O'Neill et al., 1998), 7-Cl-kinyurenic acid (CK, Wood et al., 1992) MK801 Stereoisomers of (+ MK or -MK; manufactured by Dravid et al.,) Were administered as described in the literature in various rodent or rabbit ischemia models, indicating a decrease in infarct volume (see below). The percent reduction in infarction was calculated from the ratio of infarct volume in the drug to the control for all compounds except ketamine and 7-Cl-quinuric acid to determine the percent reduction in nerve density by the drug. .
1, compounds 93-4, 93-5, 93-8, 93-31, 93-40, 93-43, 93-97, (+) MK801, (-) MK801, and all other compounds. Efficacy elevations at pH 6.9 vs. 7.6 were calculated as the number of experiments reported in Tables 1 and 2, as described above. The pH rise for iffenprodil (IF), CP 101,606 (CP) was determined from the literature (Mott et al., 1998).
Example 5: Evaluation in an In Vivo Model of Neuropathic Pain
Way
Fauna: Male Sprague-Dawley weighed at 100 ± 10 g on the day of the procedure and 250 ± 10 g on the day of the test (Hsd: Sprague-Dawley)^®TMSD^®TM, Harlan, Indianapolis, Indiana, U.S.A.), 3 per cage. The animals were free to enter and exit the food and water, and maintained a 12:12 hour day / night schedule. Animal populations were maintained at 21 ° C., 60% humidity. All experiments were performed in accordance with the guidelines of the International Association for the Study of Pain and were approved by the University of Minnesota Animal Care and the American Association.
Drug and Dose Solution:The drug was dissolved in 1% v / v DMSO and 66% v / v PEG 400 in distilled water. The compound i.p. Administered by root.
Causes of chronic neuropathic pain:Spinal nerve ligation (SNL) model (Kim and Chung 1992 Pain 50: 355-63.) Was used to cause chronic neuropathic pain. Animals were anesthetized with isoflurane and the left L5 transverse procedure was removed to tightly ligate L5 and L6 spinal cord nerves with 6-0 silk sutures. The wound was then closed with internal sutures and external staples. The wound clip was removed 10 days after the procedure.
Mechanical allodynia test: Baseline and post-treatment values for innocuous mechanical sensitivity were determined by varying stiffness (0.4) according to the up-down method (haplan, Bach et al. 1994 J Neurosci Methods 53: 55-63). , 0.7, 1.2, 2.0, 3.6, 5.5, 8.5, and 15 g) were evaluated using 8 Semmes-Weinstein filament (Stoelting, Wood Dale, IL, USA). Prior to testing, the animals were placed on perforated metal platforms and allowed to acclimate to the surroundings for at least 30 minutes. In each treatment group, the mean and standard error for the mean (SEM) for each animal were determined. Since such stimuli are generally considered not to be painful, in this test, sufficient damage-induced increase in responsiveness was interpreted as a measure of mechanical allodynia.
Experimental design: Prior to drug administration, this Frei baseline measurements were taken at 30 minutes and 24 hours, respectively. Additional Bone Frey measurements were performed at 30, 60, 120 and 240 minutes. The timeline for the test is summarized below. Experimental group: vehicle (1% DMSO + 66% PEG 400, ip, 4 ml / kg, n = 10) in 30 mg / kg Compound 93-31 test (ip, 4 ml / kg, n = 10) 100 mg / kg Compound 93-31 Test (ip, 4 ml / kg, n = 10) 30 mg / kg Compound 93-97 Test (ip, 4 ml / kg, n = 10) 100 mg / kg Compound 93 -97 test (ip, 4 ml / kg, n = 10) .100 mg / kg gabapentin (ip, 4 ml / kg, n = 12) (total rat: 62).
Experiment timetable

Blinding Process: The drug solution was administered by another experimenter who did not perform a behavioral test.
Data analysis: Statistical analysis was performed using Prism ™ 4.01 (GraphPad, San Diego, Calif., USA). Mechanical allodynia of damaged paws was determined by comparing the values observed in opposite and ipsilateral paws within the vehicle group. The stability of vehicle group damaged poe values over time was tested using the Friedman two-way analysis of variance by rank. Drug effects at each time point were analyzed by Dunn's post hoc test after Kruskal-Wallis one-way analysis of variance by rank.
result
Bone Frey Test
Testing of mechanical allodynia was initiated 14 days after SNL surgery. At baseline (30 minutes before drug administration) and at 30, 60, 120, and 240 minutes after a single drug administration, tests were performed on both injured (ipsilateral) and normal (opposite) pores.
At baseline all animals showed mechanical allodynia in injured poes (Table 2). The level of injury was comparable between the populations and significantly differed from that observed in normal paws in the vehicle treatment group throughout the bone frey threshold study of damaged paws (FIG. 6). 6 shows that animals in the vehicle group showed noticeable mechanical allodynia over the entire study period. Shown are the mean ± SEM (n = 10) Von Frey thresholds in the normal PoEs of animals treated with damaged PoEs and vehicles. The difference between the pows was noticeable at all time points (Mann-Whitney test). Compounds 93-31 and 93-97 did not affect the Von Frey thresholds measured in normal paws (FIGS. 7 and 8). FIG. 7 shows that compounds 93-31 do not change the von Frey threshold in normal paws. Shown are vehicle, gabapentin or i.p. Mean ± SEM (n = 10) Von Frey threshold in normal paws of animals treated with 30 and 100 mg / kg doses of Compound 93-31 administered. 8 shows that compound 93-97 does not change the von Frey threshold in normal paws. Shown are vehicle, gabapentin or i.p. Mean ± SEM (n = 10) Von Frey threshold in normal paws of animals treated with 30 and 100 mg / kg doses of Compound 93-97 administered.
Table 2:-Damaged Poe-Bone Frey Threshold

Table 3:-Damaged Poe-Statistical Analysis Summary

Treatment with compound 93-31 (100 mg / kg i.p.) resulted in analgesic discernible at 30 and 60 minutes after administration (FIG. 9). 9 shows i.p. of compound 93-31 (100 mg / kg). It is shown that administration reduces mechanical allodynia. Shown are vehicles, gabapentin (reference compound) or i.p. Mean ± SEM (n = 10-12) Von Frey threshold in injured poes of animals treated with 30 and 100 mg / kg doses of Compound 93-31 administered. Post-hawk analysis (Dunn test) showed significant pair-wise differences between compound 93-31 (100 mg / kg) and vehicle group at 30 and 60 minutes (p <0.01). The effects of gabapentin were also noticeable at 60, 120 and 240 minutes (p <0.001, p <0.01, p <0.01, respectively). The analgesic effects of 30 mg / kg of compound 93-31, 30 and 100 mg / kg of compound 93-97 were not present at any point in the study. Statistical analysis of the vehicle group in this study indicated that there was no noticeable difference in the Bone Frey threshold between baseline and the 30, 60, 120 and 240 minute time points (Friedman two-way ANOVA).
In addition, i.p. Compounds 93-97 administered did not relieve mechanical allodynia in SNL rats. 10 shows i.p. of compound 93-97 (30 and 100 mg / kg). Dosing does not affect the Von Frey threshold. Shown are vehicles, gabapentin (reference compound) or i.p. Mean ± SEM (n = 10-12) von Frey Threshold in injured paws of animals treated with 30 and 100 mg / kg doses of Compound 93-97 administered. The effects of gabapentin were also noticeable at 60, 120 and 240 minutes (p <0.001, p <0.01, p <0.01, respectively).
Some adverse events were observed in the animals tested in this study (8 of 62 animals). The observed side effects were riding and stretching (8 observation groups). These side effects are i.p. Most commonly observed for the first few minutes (~ 5 minutes) after drug administration. Stretching / riding was observed in all study groups including the animal group treated with vehicle i.p. (3/10) and the animal group showing no dependence on drug dose. The severity of these side effects was not severe and did not interfere with endpoint measurement sufficient to exclude animals from the study. Table 1 summarizes the side effects observed in this study. Some side effects were observed during endpoint measurement. The most common was stretching / ride, which could be a sign of some visceral pain or irritability. This is the vehicle and i.p. Observed in the drug treatment group. Vehicle i.p. in a subset of the fauna. It was thought to be associated with administration. It was relatively sparse, short-lived (<5 minutes), and not large enough to interfere with endpoint measurement.
Table 1: Side Effects

Compound 93-31 showed i.p. at 100 mg / kg. When administered, it has been shown to relieve mechanical allodynia in the SNL model of neuropathic pain. Compound 93-97 could not relieve mechanical allodynia in SNL rats at the doses tested in this study (30 and 100 mg / kg). Compound 93-31 (100 mg / kg) initiated action faster than the reference compound gabapentin (100 mg / kg) (30 minutes) and exhibited a shorter duration of action (60 minutes). The peak threshold observed in the group of animals treated with the 100 mg / kg dose of Compound 93-31 was approximately half that observed in the normal poe. If full reversal can be performed with higher doses of compound 93-31, this suggests that the ED 50 is approximately 100 mg / kg.
Example 6: pH dependence of selected compounds
A series of n-alkyl derivatives was tested for pH dependence.

Example 7: Xenopus oocyte IC for antagonism of NMDA-NR2B containing receptors in assays ₅₀ In rats versus rat receptor cDNA
In the SNL model of neuropathic pain of the above examples using rat NMDA and human NMDA receptors to compare the potency increase, the efficacy of several compounds at pH 6.9 and 7.6 was evaluated according to ex vivo screening and testing methods. Determination of pH dependent potentiation in action is determined by IC for blocking of receptors at pH 6.9.₅₀IC for blocking of receptors at pH 7.6₅₀Divided by. The increase in pH dependent potency obtained from the rat receptor did not predict the rise in pH dependent potency obtained for the human receptor for all compounds.
Table A: Rat vs. Human pH Efficacy Elevation

Although the above-described invention has been described in detail for the purpose of clarity and understanding, it will be apparent to those skilled in the art that various changes may be made in form and detail without departing from the true scope of the invention.

Claims (26)

In cells expressing human NMDA receptors, identifying compounds useful for treating or preventing pH-lowering diseases in the area of infected tissue, comprising assessing the potency difference of the compounds at disease-induced and physiological pH. How to. The compound of claim 1 wherein the potency difference is at least 5 times the IC ₅₀ of the compound at physiological pH and disease induced pH until the 95% confidence interval for potency boost does not change by at least 15% with the addition of a new experiment. Method evaluated by repeated measurement. The method of claim 1, wherein the difference in potency is an increase in potency at disease induced pH as compared to potency at physiological pH. The method of claim 1, wherein the difference in potency is an increase in potency at disease induced pH relative to physiological pH. The method of claim 1, wherein the disease of lowering pH in the area of infected tissue is neuropathic pain, ischemia, Parkinson's disease, epilepsy and traumatic brain injury. ) Selected from the group consisting of The method of claim 4, further comprising identifying a compound having at least 5 potency boosts in cells expressing a human NMDA receptor. The compound of claim 4, wherein the potency increase of the compound in cells expressing a human NMDA receptor is at least two greater than the potency boost of the same compound when tested in cells expressing a non-human NMDA receptor. Further comprising the step of identifying. 8. The method of claim 7, wherein the extra-human NMDA receptor is a rat NMDA receptor. The method of claim 1, wherein the infected tissue is selected from the group consisting of brain tissue, tissue damaged by ischemia, tissue affected by pain, tissue affected by neuropathic pain, and tissue affected by traumatic brain injury. . The method of claim 1, wherein the 95% confidence interval does not change more than 10% with the addition of a new experiment. The method of claim 1, wherein the 95% confidence interval does not change more than 5% with the addition of a new experiment. The method of claim 1, wherein the efficacy difference experiment is repeated five times. (i) assessing the elevated potency of a compound that inhibits human NMDA receptors at disease induced and physiological pH in cells expressing human NMDA receptors;
(ii) testing the compound in vivo and measuring the effect of the compound on the pain threshold;
(iii) selecting a compound associated with an increase in at least 5 efficacy in step (i) and at least a 2 fold increase in pain threshold in step (ii),
A method of identifying a compound useful for treating or preventing a pain disorder in an infected tissue area. The method of claim 13, wherein the potency increase is determined by at least five repeated measurements of the IC ₅₀ of the compound at physiological pH and disease induced pH until the 95% confidence interval for the difference in potency does not change by at least 15% in addition to a new experiment. , How measured. The method of claim 14, wherein the potency boost is measured at least 12 times. The method of claim 13, wherein the pain threshold is measured until the 95% confidence interval does not change by more than 5% with the addition of a new experiment. The method of claim 16, wherein the pain threshold is measured at least 12 times. The method of claim 13, wherein the 95% confidence interval of the efficacy gain obtained in step (i) does not change by more than 15%. The method of claim 13, wherein the 95% confidence interval of the efficacy gain obtained in step (i) does not change more than 5%. The method of claim 13, wherein the potency boost experiment of step (i) is repeated at least five times. The method of claim 13, wherein step (ii) comprises testing the compound in an animal model of neuropathic pain. The method of claim 13, wherein the 95% confidence interval of the pain threshold obtained in step (ii) does not change by more than 15%. The method of claim 13, wherein the 95% confidence interval of the pain threshold obtained in step (ii) does not change more than 5%. The method of claim 13, wherein the pain disorder lowers the pH in the affected tissue. The method of claim 13, wherein the disease that lowers pH in the affected tissue area is neuropathic pain. (i) In cells expressing human NMDA receptors, the compound's efficacy increase at disease induced and physiological pH was repeated at least five times until the 95% confidence interval did not change more than 15% with the addition of a new experiment. Evaluating by;
(ii) Test the compound in an animal model of neuropathic pain and repeat the test at least 12 times until the 95% confidence interval did not change by more than 5% with the addition of a new experiment. Measuring;
(iii) selecting at least five compounds of potency increase in step (i), and in step (ii) selecting a compound that is associated with at least a two-fold increase in pain threshold,
A method of identifying compounds useful for treating or preventing neuropathic pain.

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