The central nervous system (CNS) is composed of the brain contained in the cranium, and the medulla spinalis or spinal cord in the vertebral canal. The brain contains 12 cranial nerves. The spinal nerves spring from the medulla spinalis, and are transmitted through the intervertebral foramina. The spinal cord, which originates immediately below the brain stem, extends to the first lumbar vertebra, designated L1. Beyond L1 the spinal cord becomes the cauda equina. The spinal cord is comprised of 31 pairs of nerves comprising 8 pairs of cervical spinal nerves, 12 pairs of thoracic spinal nerves, 5 pairs of lumbar spinal nerves, 5 pairs of sacral spinal nerves, and 1 pair of coccygeal spinal nerves. The first cervical nerve (called the suboccipital nerve) emerges from the vertebral canal between the occipital bone and the atlas; the eighth issues between the seventh cervical and first thoracic vertebra. Each nerve is attached to the medulla spinalis by two roots, an anterior or ventral, and a posterior or dorsal, the latter being characterized by the presence of a ganglion, the spinal ganglion. Each spinal nerve is formed from a ventral or anterior root comprising axons from motor neurons and a dorsal or posterior root comprising nerve fibers from sensory neurons. The anterior root (radix anterior; ventral root) emerges from the anterior surface of the medulla spinalis as a number of rootlets or filaments (fila radicularia), which coalesce to form two bundles near the intervertebral foramen. The posterior root (radix posterior; dorsal root) is larger than the anterior due to the greater size and number of its rootlets which are attached along the posterolateral furrow of the medulla spinalis and unite to form two bundles which join the spinal ganglion. The posterior root of the first cervical nerve is smaller than the anterior. The spinal ganglia (ganglion spinale) are collections of nerve cells on the posterior roots of the spinal nerves. Each ganglion is oval in shape, reddish in color, and its size is in proportion to that of the nerve root on which it is situated. It is bifid medially where it is joined by the two bundles of the posterior nerve root. The ganglia are usually but not always found in the intervertebral foramina, immediately outside the points where the nerve roots perforate the dura mater. The ganglia of the first and second cervical nerves lie on the vertebral arches of the atlas and axis respectively, the ganglia of the sacral nerves are inside the vertebral canal, while the ganglion on the posterior root of the coccygeal nerve lies within the sheath of dura mater. When a dorsal root and a ventral root unite, they form a spinal nerve providing both motor and sensory utility. Spinal nerves split into two main branches, the dorsal ramus which innervates the skin of the back and deep back muscles, and the ventral ramus which innervates everything else from the neck inferiorly and also forms nerve plexuses which are a network of converging and/or diverging nerve fibers. The ganglia are comprised of unipolar nerve cells, and from these the fibers of the posterior root originate. Two other forms of cells are also present, i.e., the cells of Dogiel, whose axons ramify close to the cell (type II, of Golgi), and which are distributed entirely within the ganglion; and multipolar cells similar to those found in the sympathetic ganglia. The ganglia of the first cervical nerve may be absent, while small aberrant ganglia consisting of groups of nerve cells are sometimes found on the posterior roots between the spinal ganglia and the medulla spinalis. Each nerve root has a covering comprising pia mater, and is loosely invested by the arachnoid, the latter being prolonged as far as the points where the roots pierce the dura mater. The two roots pierce the dura mater separately, each receiving a sheath from this membrane; where the roots join to form the spinal nerve this sheath is continuous with the epineurium of the nerve.
The largest nerve roots and the largest spinal nerves are those of the lower lumbar and upper sacral nerves, and are attached to the cervical and lumbar swellings of the medulla spinalis.
These nerves are distributed to the upper and lower limbs. Their individual filaments are the most numerous of all the spinal nerves. The roots of the coccygeal nerve are the smallest. Immediately beyond the spinal ganglion, the anterior and posterior nerve roots unite to form the spinal nerve which emerges through the intervertebral foramen. Each spinal nerve receives a branch (gray ramus communicans) from the adjacent ganglion of the sympathetic trunk, while the thoracic and the first and second lumbar nerves each contribute a branch (white ramus communicans) to the adjoining sympathetic ganglion. The second, third, and fourth sacral nerves also supply white rami. These are not connected with the ganglia of the sympathetic trunk, but run directly into the pelvic plexuses of the sympathetic nerve system. A typical spinal nerve contains fibers belonging to two systems, i.e., the somatic nerve system, and the sympathetic or splanchnic nerve system, together with nerve fibers connecting these systems with each other.
The somatic fibers are efferent and afferent. The efferent fibers originate in the cells of the anterior column of the medulla spinalis, and run outward through the anterior nerve roots to the spinal nerve. They convey impulses to the voluntary muscles, and are continuous from their origin to their peripheral distribution. The afferent fibers convey impressions inward, for example from the skin, and originate in the unipolar nerve cells of the spinal ganglia. The single processes of these cells divide into peripheral and central fibers, and the latter enter the medulla spinalis through the posterior nerve roots.
The sympathetic fibers are also efferent and afferent. The efferent fibers, preganglionic fibers, originate in the lateral column of the medulla spinalis, and are conveyed through the anterior nerve root and the white ramus communicans to the corresponding ganglion of the sympathetic trunk where they may end by forming synapses around its cells, or may run through the ganglion to end in another of the ganglia of the sympathetic trunk, or in a more distally placed ganglion in one of the sympathetic plexuses. They end by forming synapses around other nerve cells. From the cells of the ganglia of the sympathetic trunk other fibers, postganglionic fibers, take origin; some of these run through the gray rami communicantes to join the spinal nerves, along which they are carried to the blood vessels of the trunk and limbs, while others pass to the viscera, either directly or after interruption in one of the distal ganglia. The afferent fibers are derived partly from the unipolar cells and partly from the multipolar cells of the spinal ganglia. Their peripheral processes are carried through the white rami communicantes, and after passing through one or more sympathetic ganglia without interruption end in the tissues of the viscera. The central processes of the unipolar cells enter the medulla spinalis through the posterior nerve root and form synapses around either somatic or sympathetic efferent neurons to complete reflex arcs. The dendrites of the multipolar nerve cells form synapses around the cells of type II (cells of Dogiel) in the spinal ganglia. By this path an original impulse is transferred from the sympathetic to the somatic system, through which it is conveyed to the sensorium.
After emerging from the intervertebral foramen, each spinal nerve gives off a small meningeal branch which reenters the vertebral canal through the intervertebral foramen and supplies the vertebra and their ligaments as well as the blood vessels of the medulla spinalis and its membranes. The spinal nerve then splits into a posterior or dorsal, and an anterior or ventral division, each receiving fibers from both nerve roots.
The spinal cord provides a means of motor and sensory communication between the brain and the nerves of the peripheral nervous system (PNS) which includes the somatic nervous system (SNS) and the autonomic nervous system (ANS). The somatic nervous system is voluntary, includes the nerves serving the musculoskeletal system and the skin, and reacts to outside stimuli affecting the body. The autonomic nervous system is involuntary, and controls and maintains homeostasis or normal function. The autonomic nervous system is comprised of the sympathetic nervous system associated with the flight or fight response, and the parasympathetic nervous system responsible for maintaining regular life functions such as heartbeat and breathing during normal activity. Nerve roots pass out of the spinal canal through the intervertebral foramen and distribute to the body anteriorly for motor activity or posteriorly for sensory activity. The anterior nerve divisions supply the front of the spine including the limbs while the posterior nerve divisions are distributed to the muscles behind the spine.
Cerebrospinal fluid is secreted from the choroids plexus in the brain and is present in the brain ventricles, in the spinal canal, and in the spinal cord. The fluid circulates among these tissues and cushions the tissues in the CNS from the effects of traumatic injury. The CNS in a normal adult contains about 150 milliliters of cerebrospinal fluid.
The brain and spinal cord are covered and protected by meninges membranes comprising strong connective dura mater tissue, or dura, as a gray outer layer of the spinal cord and nerve roots; arachnoid mater, thinner than the dura mater; and pia mater, the innermost layer covering the nerves as a delicate and highly vascular membrane providing blood to the neural structures.
The dura membrane that covers the spine and nerve roots in the neck is surrounded by the epidural space. Nerves pass through the epidural space to the neck, shoulder and arms. Inflammation of these nerve roots may cause pain in these regions due to irritation from a damaged disc or from contract with the bony structure of the spine.
Injury to the nerves in the central nervous system such as nerves in the spinal cord can result in functional impairment which can take the form of permanent loss of sensation and paraplegia. Most of the deficits associated with spinal cord injury such as traumatic spinal cord injury, crush injury, or lesion in the spinal nerves result from cell death and the loss of axons in the spinal neuronal population that are damaged in the central nervous system (CNS) which is comprised of nerves in the spinal cord and brain. Axons do not otherwise regrow across a lesion site in a damaged nerve in the CNS. Neurodegenerative diseases of the CNS are also associated with cell death and axonal loss. Representative diseases of the CNS include those that can result in impairment include stroke, human immunodeficiency virus (HIV) dementia, prion diseases, Parkinson's disease, Alzheimer's disease, multiple sclerosis, traumatic brain injury, and glaucoma. The ability to stimulate growth of axons from the injured, damaged, diseased or otherwise affected neuronal population would improve recovery of lost neurological functions, and protection from cell death can limit the extent of damage in the CNS. For example, following a white matter stroke, axons are damaged and lost, even though the neuronal cell bodies are alive, and stroke in gray matter kills many neurons and non-neuronal (glial) cells.
Effective neuroprotective and neuroregenerative agents are desirable to potentially limit damage and to induce repair to the CNS. Compounds which promote axon growth in nerve cells of the CNS are especially desirable for treatment of damaged, injured, and/or diseased nerves in the CNS. Compounds which promote axon growth in nerve cells of the peripheral nervous system (the PNS) are also especially desirable for treatment of damaged, injured, and/or diseased nerves in the PNS.
Traumatic injury of the spinal cord can result in permanent functional impairment. Axon regeneration in nerve cells of the CNS does not occur at the site of injury or lesion site in the mammalian CNS because growth inhibitory proteins present in anatomical structures immediately proximal to nerves as substrate-bound proteins block axon growth. Anatomical structures such as myelin sheaths immediately proximal to nerves are referred to as inhibitory substrates. While compounds such as trophic factors can enhance neuronal differentiation and stimulate axon growth in tissue culture, most factors and compounds that enhance growth and differentiation cannot promote axon regenerative growth on inhibitory substrates.
A compound which stimulates axon growth in tissue cell culture and induces axon growth on growth inhibitory substrates is potentially useful for therapeutic use in axon regeneration when applied to cells residing in the CNS, such as directly to the site of a lesion in the CNS. Trophic and differentiation factors that stimulate growth on permissive substrates, that is, in the absence of inhibitory proteins or in the absence of inhibitory substrates, in tissue culture include neurotrophins such as nerve growth factor (NGF) and brain-derived growth factor (BDNF). Neither NGF nor BDNF promotes neurite growth or axon regeneration in nerve cells on inhibitory substrates, and neither is effective in promoting axon regeneration in nerve cells in the CNS in vivo.
Cell death can occur by two major mechanisms, necrosis and apoptosis. While necrotic cell death results in cell lysis and release of cell contents, cellular apoptosis is programmed cell death that results in the relatively tidy packaging of cells which die with the prevention of release of cellular contents. Apoptosis is characterized morphologically by cell shrinkage, nuclear pyknosis, chromatin condensation, and blebbing of the plasma membrane. Traumatic injury and ischemia can lead to apoptosis of both neurons and non-neuronal cells, and this cell death is responsible for functional deficits after injury or ischemia. A cascade of molecular and biochemical events is associated with apoptosis including activation of an endogenous endonuclease that cleaves DNA into oligonucleosomes detectable as a ladder of DNA fragments in agarose gels. Apoptotic endonucleases not only affect cellular DNA by producing the classical DNA ladder but also generate free 3′-OH groups at the deoxyribose ends of these DNA fragments. A technique called Tunel labeling labels DNA fragments as a means to detect apoptotic cells.
Rho kinase, an enzyme that resides in the interior of cells such as nerve cells, is a target for treatment of cancer, metastasis, and hypertension. Rho kinase inhibitors may be useful to treat eye diseases such as glaucoma, as well as to inhibit cancer cell migration and metastasis.
Rho kinase antagonists may be useful in treatment of hypertension, asthma, and vascular disease such as thrombosis.
The superfamily of small GTP-binding proteins or small G-proteins can be divided according to the similarity of amino acid sequences into 5 groups of Ras, Rho (short for Ras homologue), Rab, Arf and others. The small GTP-binding proteins have a molecular weight of 20,000-30,000 Daltons, specifically bind GDP and GTP, and exhibit GTPase activity by hydrolyzing bound GTP. Rho is specifically ADP ribosylated and inactivated by C3, a botulinum toxin.
Protein kinases are proteins that phosphorylate and control the activity of other proteins by transferring a phosphate from ATP (adenosine triphosphate) to an amino acid, e.g. serine, threonine, or tyrosine, on another (target) protein. Target proteins regulated by phosphorylation include enzymes that transduce signals in a cellular environment and enzymes that turn certain genes on or off and can thereby regulate progression of many different diseases.
Rho kinase regulates cell cytoskeleton organization, cell adhesion and cell motility, and a cell's cycle. Rho kinase is a serine/threonine kinase, and is a Rho binding protein or an effector of Rho which is a GTPase, which catalyzes the reaction: GTP (Guanosine 5′-triphosphate)+H2O (water) to GDP (Guanosine 5′-diphosphate)+phosphate ion, and is linked to a cell membrane in its active state. Inhibition of Rho kinase by a compound of this invention can have potential therapeutic applications in a mammal, such as reduction of tumor metastasis in cancer, relaxation of vascular tension in cardiovascular disease, and reduction of ocular pressure in glaucoma, among other applications. Two different Rho kinase inhibitors, Fasudil™ and Radicut™, are in use in humans for treatment of stroke. Rho kinase is called ROK (Rho-associated kinase) and ROCK (Rho associated Rho kinase), ROKa, or ROCKI. Isoforms of ROCK exist: ROCKII (has 64% sequence identity with ROCKI, and ROKβ is 90% identical. The protein has three important domains: a Rho-binding domain (RB), a C-terminal PH (Pleckstrin homology) domain, and a catalytic domain.
ROCKI and ROCKII are each activated by Rho. An important distinction between ROCKI and ROCKII comprises their respective in vivo tissue distributions in a mammal (e.g., see Nakagawa et al., FEBS Lett. 1996 Aug. 26; 392(2):189-93). Analysis of relevant mRNA concentration levels has shown that ROCKI has widespread tissue distribution, but that there is relatively little ROCKI in brain and in skeletal muscle. Expression of ROCKII is high in brain, heart and lung, but is relatively low in liver, stomach, spleen, kidney and testis. Western blots used to study protein concentration levels show ROCKII concentration levels to be low in liver and kidney (Wibberley et al. 2003, British J. Pharmacology 138:757-766), whereas ROCKII is highly expressed in brain as compared to ROCKI (Leemhuis et al. 2002, J Pharmacol Exp Ther. 300:1000). Therefore, for therapeutic and diagnostic use in the CNS of a mammal, an inhibitor that is specific for inhibition of ROCKII would be desirable.
In this regard, known Rho kinase inhibitor Y27632 [(R)-(+)-trans-4-(1-aminoethyl)-N-(4-pyridyl)cyclohexane-carboxamide as a dihydrochloride salt] inactivates (inhibits) both ROCKI and ROCKII (Ishizaki et al. 2000, Molecular Pharmacology 57:976).
In addition, a Rho kinase inhibitor that is in clinical use for treatment of stroke, Radicut™ (edaravone; 3-methyl-1-phenyl-2-pyrazoline-5-one; 5-methyl-2-phenyl-2,4-dihydro-3H-pyrazol-3-one), is known to have kidney toxicity. An estimated 180,000 patients have taken the drug Radicut™ since it was approved for use in 2001 in Japan. Ninety-three were later struck down with kidney failure and 40 died. A Rho kinase inhibitor specific for ROCKII may not be expected to show this adverse effect profile in part because of the low abundance of ROCKII in liver and kidney (Wibberley et al. 2003, British J. Pharmacology 138:757-766).
Nearly all protein kinase inhibitors that have been developed are ATP-competitive, and for this reason the “drug concentration required for 50% inhibition” (IC50) of a protein kinase depends on the concentration of ATP used in the assays. A drug concentration required to suppress phosphorylation of a target substrate in a cell can be higher than the drug concentration required for inhibition of a protein kinase in vitro.
A compound known to inhibit the activity of Rho kinases is (R)-trans-4-(ethan-1′-amino)-N-(4″-pyridyl)cyclohexane carboxamide dihydrochloride known as Y-27632, which is available from Sigma Chemical Company and from Calbiochem in powder form and which can be dissolved in DMSO (dimethyl sulfoxide, Sigma) and which can be useful as a reference Rho kinase inhibiting compound.
Rho kinase regulates axon growth and regeneration in nerve cells, cell motility and metastasis, smooth muscle contraction, and apoptosis, and is a target for therapeutic treatment in many disease applications, including repair in the central nervous system. Rho kinase is activated by Rho and Rho kinase inhibitors block Rho signaling. Mutations of Rho family regulatory proteins have been found in clinical oncology samples which suggests that perturbation or alteration or interruption of, or interference with, the Rho signaling pathway can be a useful therapeutic modality. Examples with specificity for Rho include the DLC1 gene in hepatocellular carcinoma, p-190-A, which is in a region that is altered in gliomas and astrocytomas, GRAF, which has loss of function mutations in leukemia, and LARG, which is found in some gene fusions found in acute myeloid leukemia. Genetically engineered point mutations can activate RhoA and induce cellular transformation in vitro.
Rho kinase inhibitors have widespread potential for use in the treatment of neurodegenerative diseases, particularly if the Rho kinase inhibitors have the property to enhance plasticity and axon regeneration in neurons. However, there is much scientific evidence for a direct link between Rho signaling and neurodegenerative disease. In an animal model of Alzheimer's Disease (AD), there is clear evidence that Rho kinase inhibitors can reduce the pathological hallmarks of the disease.
A trans-4-amino(alkyl)-1-pyridylcarbamoylcyclohexane, designated as Y27632, is a Rho kinase inhibitor and available from Calbiochem. Y27632 is described in U.S. Pat. No. 4,997,834, the entire content of which is incorporated herein by reference. Y27632 has been used to demonstrate that inhibition of Rho kinase is effective in preventing metastasis. Other compounds are described in U.S. Pat. No. 5,478,838, the entire content of which is incorporated herein by reference.
Rho signaling antagonists can be useful in treatment of hypertension. For example, Y-27632 can relax smooth muscle and increase vascular blood flow. Y-27632 is a small molecule that can enter cells, and is not toxic in rats after oral administration of 30 mg/kg for 10 days. Y-27632 reduces blood pressure in hypertensive rats, but does not affect blood pressure in normal rats.
A number of Rho kinase inhibitors are known. For example, the compound NHM-1152 can act as vascular relaxant in vascular vasospasm. The compound known as hydroxy fasudil may find use in the treatment of stroke after intravenous application and can reduce infarct volume, improve outcomes, can have anti-ischemic properties in vasospastic angina, and inhibits neutrophil migration in ischemic brain. A fasudil compound called HA-1077 is an antivasospasm agent which improves cerebral hemodynamic activity, inhibits production of superoxide anion by neurotrophils, and may find use in the treatment of spinal cord injury, stroke, subarachnoid hemorrhage, and cerebral infarction. U.S. Pat. No. 6,218,410, the disclosure of which is hereby incorporated by reference in its entirety, describes a Rho kinase inhibitor. U.S. patent application Ser. No. 10/022,301 (McKerracher et al) published as U.S. 2002/0119140, the disclosure of which is hereby incorporated by reference in its entirety, describes Rho family antagonists and their use to block inhibition of neurite outgrowth. Canadian Patent applications 2,304,981 (McKerracher et al) and 2,325,842 (McKerracher), the disclosure of each of which is hereby incorporated by reference in its entirety, disclose the use of Rho antagonists such as for example C3 and chimeric C3 proteins as well as substances selected from among known trans-4-amino(alkyl)-1-pyridyl-carbamoylcyclohexane compounds or Rho kinase inhibitors for use in the regeneration of axons. C3 inactivates Rho by ADP-ribosylation and can be relatively non-toxic to nerve cells at therapeutically effective doses. U.S. Pat. Nos. 4,857,301, and 5,741,792, the disclosure of each of which is hereby incorporated by reference in its entirety, describes a number of 4-substituted piperidine compounds. U.S. Pat. No. 6,140,333, the disclosure of which is hereby incorporated by reference in its entirety, describes certain piperidine compounds, one of which is a 4-aminomethylpiperidine acylated at the piperidine ring nitrogen by a group containing a carbon adjacent to the carbonyl group to which carbon is attached an aryl group and a hydroxyl group. U.S. Pat. No. 6,020,352, the disclosure of which is hereby incorporated by reference in its entirety, describes the use of certain 1-phenyl-2-piperidinoalkanol derivatives to treat ischemic disorders of the retina and optic nerve.
U.S. Pat. Nos. 4,849,521, 4,584,303, 4,866,077, 4,933,353, and 6,169,097, the disclosure of each of which is hereby incorporated by reference in its entirety, disclose methods to prepare a number of piperidines with substituents attached to one or more of the ring carbon atoms of a piperidine. U.S. Pat. No. 6,545,022, the disclosure of which is hereby incorporated by reference in its entirety, describes a method to prepare certain 4-substituted-4-aminoalkylpiperidines including certain 4-substitutedmethylene-4-aminomethylpiperidines.