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Experimental Therapies of Spinal Cord Injury
Experimental Therapies of Spinal Cord Injury
Wise Young, Ph.D., M.D
Rutgers University, Piscatawy, NJ 08854-8082
Started 24 December 2001
Last updated 7 January 2002
A decade ago, there were virtually no therapies for spinal cord injury other than prevention of further damage shortly after injury. In 1990, methylprednisolone was shown to improve functional recovery when given within 8 hours after injury. It stops some of the tissue inflammatory and free radical responses that contribute to progressive tissue loss. In the 1990's, major advances in medical, surgical, and rehabilitative technology significantly improved the quality of life of people with spinal cord injury. These include better urological and orthopedic procedures, as well as improved drug therapies for spasticity and neuropathic pain, fertility, and sexual function.
For many years, most scientists believed that restoration of function requires regeneration. However, much data suggest that several non-regenerative therapies, such as decompressive and untethering surgery, can produce functional recovery. Likewise, intensive forced-use training can restore function but through reversal of learned non-use even long times after injury. Finally, several therapies, such as 4-aminopyridine and remyelination with oligodendroglia and Schwann cells, may improve function of existing connections.
For those with insufficient axons for function, regeneration is necessary. The good news is that less than 10% of the spinal cord is necessary and sufficient to support substantial recovery in spinal cord injury, explaining why many people with so-called "incomplete" spinal cord injuries can recover substantially. The bad news is that the axons have to grow from the injury site to innervate neurons far above or below the injury site.
Dozens of experimental therapies have been reported to restore function in animal models of spinal cord injury. The profusion of therapies for spinal cord injury is confusing not only for families with spinal cord injury community but for clinicians and scientists as well. It is helpful to put some of the therapies into perspective by categorizing them by mechanism or approach. Therapies that are already in clinical trial or clinical use are highlighted in red. I have placed asterisks next to therapies that have been in clinical trial (*), are undergoing clinical trial (**), or are already in clinical use (***).
Experimental treatments for spinal cord injury fall into seven categories: neuroprotective, neuroreparative, neurotrophic, neuroregenerative, neurorestorative, neuroconstructive, and neurogenetic. Each of these will be discussed in turn, with key examples of therapies that may be ready to go to clinical trial. In the coming months, I shall update this list, adding references and links to clinical trial information, prioritizing the list and adding comments.
1. Neuroprotective therapies (acute) prevent further damage to the spinal cord. Many drugs have been reported to be neuroprotective but methylprednisolone was and is the first and still only treatment that was shown to prevent progressive damage in human spinal cord injury. It must be given within 8 hours after injury and for 24-48 hours because many of its actions counter some of the natural neuroreparative mechanisms if the drug is continued longer. Some of these therapies aim directly at preventing necrosis (mechanical or calcium-mediated injuries) while others aim at preventing apoptosis (gene-mediated programmed cell death).
- Glucocorticoids. Glucocorticoids are steroid hormones that are released by injury and stress. They curb inflammatory responses. They are frequently used to treat inflammatory disorders of the brain and spinal cord.
- Methylprednisolone*. This is a glucocorticoid steroid that improves neurological recovery when given within 8 hours after injury and for only 24-48 hours. It is given in much higher doses (30 mg/kg) than is usually used for anti-inflammatory purposes and lower doses are not effective. The drug also should not be started later than 8 hours after injury because it is not only ineffective but potentially deleterious. At least four clinical trials have shown improved recovery but there remains considerable controversy concerning its efficacy. It is nevertheless the current standard clinical therapy in the United States and many other countries, not only for spinal cord injury but for multiple sclerosis as well.
- Tetracyclines. These are antibiotics that have long been used to treat bacterial infections. However, several tetracyclines appear to also change the gene expression of mammalian cells.
- Minocycline. This is an antibiotic of the tetracycline class. This antibiotic, however, appears to have antioxidant and free radical scavenging capabilities, as well as anti-inflammatory activities that are still not well understood. Minocycline was approved by the FDA in 1996 for the treatment of rheumatoid arthritis, in addition to its well known effects as a potent antibiotic for urinary tract infections. Minocycline has been reported to inhibit expression of metalloproteases, endogenous enzymes that break down extracellular matrix proteins (see posting in the Research Forum). In November 2001, Barry Festoff and his colleagues reported at the Society of Neuroscience meeting that minocycline has dramatic neuroprotective effects on contused rat spinal cords. This is still a preliminary report and has not yet been confirmed.
- Glutamate receptor blockers. Glutamate is an amino acid and an excitatory neurotransmitter responsible for a wide variety of functions, including pain transmission in the brain and spinal cord. Glutamate receptors come in two varieties: NMDA and AMPA. NMDA receptors open calcium channels while AMPA receptors turn on a variety of intracellular messengers that modulate other functions of cells. Both receptors can have profound effects on neuronal and glial function.
- MK801. This is one of the first NMDA receptor blockers that was widely studied. Over the past two decades, MK801 has been reported to be neuroprotective in head injury, stroke, and spinal cord injury. However, to date, none of the clinical trials of NMDA receptor blockers have shown positive results. MK801 was withdrawn from clinical development by Merck several years ago because drug may cause neuronal death in animals.
- Agmantine. This is an AMPA receptor blocker that was recently reported by Yezierski, et al. to reduce neuropathic pain and also progressive tissue damage in rat spinal cord injury models. To my knowledge, agmantine has not been used in clinical trials for spinal cord injury.
- GABA receptor antogonists. GABA is one of two main inhibitory neurotransmitters in the brain. The other is glycine. There are two basic forms of GABA receptors: GABA-A and GABA-B receptors. GABA-A receptors gate chloride channels and, at least in adult neurons, causes hyperpolarization and therefore inhibition of neuronal activity. GABA-B receptors act through intracellular messengers that tend to modulate other receptors. Many drugs inhibit GABA receptors, including drugs of the benzodiazepine class (valium, etc.). In addition, several hormones and natural molecules affect GABA receptors.
- Pregnenolone. This is a precursor to the hormone progesterone. Made from cholesterol by the brain, this molecule is present in high concentrations in the brain and spinal cord. Its function is not well understood but because it is made by the brain, it is sometimes called a "neurosteroid". Several studies have reported that pregnenolone (and progesterone itself) blocks GABA receptors and may be neuroprotective.
- Glutamine synthetase. This is the enzyme in that breaks down glutamate and converts it to glutamine. Present in neurons and glia, this enzyme is a key component of the metabolism of glutamate. Recently (unpublished), we discovered that this enzyme blocks GABA receptors and is neuroprotective.
- Cycloxygenase blockers. Cycloxgenase (COX) are enzymes responsible for the production of prostaglandins (PG). The PG's are an important component of inflammation, responsible for many of the reactions of tissue of inflammation, including vasodilation, swelling, local pain, blood clotting, as well as fever. Some COX blockers have been used for centuries, i.e. aspirin, to prevent inflammation. However, these drugs have serious side effects, such as ulcers and anti-coagulation. Most of these side-effects are mediated by COX-1 enzymes whereas the pain and swelling responses appear to be mediated by COX-2 receptors.
- Indomethacin, ibuprofen. These drugs are general COX inhibitors, like aspirin. Although they have never been reported to be neuroprotective in spinal cord injury models, there has been a long-standing interest in these drugs because of their potential to block prostaglandin production. However,
- COX-2 inhibitors. In the last few years, specific drugs have been designed to block COX-2 selectively. These drugs are commonly used today to treat osteoarthritis and rheumatic arthritis, as well as inflammatory pain in general. Brand names of such drugs include Vioxx and Celebrex. None of these drugs have been tested extensively for neuroprotective effects.
- Acetaminophen. This drug (better known by its brandname Tylenol) apparently blocks prostaglandin E2 (PGE2) synthetase in the central nervous system. PGE2 mediates fever and other CNS inflammation. For this reason, there is interest in the use of this drug during the acute phase of spinal cord injury, to modulate the inflammatory response.
2. Neuroreparative therapies (subacute). These therapies facilitate and promote repair of the spinal cord during the weeks that follow spinal cord injury. The mechanisms are not well understood but several therapies have been reported to accelerate and improve recovery. Many of these may stimulate cells to engage in repair and clean up. Some of these may involve antibodies.
- Protein kinase modulators
- Monosialic ganglioside (GM1)*. This molecule is a glycolipid that is present in the brain. Long been known to stimulate neuronal growth in culture, GM1 was used in Italy for several years to treat neurodegenerative disorders. In 1991, Geisler, et al. reported that GM1 improved locomotor recovery in patients with spinal cord injury. The treatment (100 mg per day intramuscular injection) was started within 48 hours after injury and continued daily for 6 weeks. A second clnical trial was started in 1993 and suggested a strong trend for beneficial effects on recovery although the trial did not meet its original objectives of therapeutic efficacy. The final report of the trial results was just recently published in http://www.spine.org. For more detailed commentary on the results, go to the Research section. A pdf copy of the reprint can be downloaded from here.
- Inflammatory cells
- Activated macrophages**. In 1999, Michal Schwartz and colleagues at the Weizmann Institute reported the blood macrophages activated by exposure to peripheral nerves and then transplanted to the spinal cord reduce tissue damage and improve neurological recovery in rats. This treatment is currently in phase 1 clinical trials in Israel and in Belgium. This treatment is apparently beneficial when applied within 2 weeks after injury but there has not been systematic testing of activated macrophages in chronic spinal cord injury.
- Activated t-lymphocytes. In 2001, Schwartz, et al. discovered that lymphocytes when activated by myelin-basic protein, and then transplanted back into the spinal cord or even injected intravascularly can significantly reduce tissue damage in the spinal cord. This treatment is apparently beneficial only when applied within 2 weeks after injury but again I don't think that the treatment has been systematically tested in chronic spinal cord injury models.
- Lymphocyte activators
- Myelin basic protein (MBP). Scientists have long used MBP, a component of myelin to initiate autoimmune responses in animals, to create models of multiple sclerosis. If animals are inoculated with MBP along with a strong inflammatory stimulator, it can cause auto-immune disease where the animals' own immune system attacks the myelin and cause demyelination. However, when the MBP is inoculated with a mild inflammatory stimulator, it apparently can activate the lymphocyte in a beneficial way.
- Glatiramer Acetate (Copaxone)*. This is a drug that has been approved for use in multiple sclerosis by the U.S. FDA. It significantly reduces the incidence and severity of exacerbating attacks. The mechanism of its action is unclear but one theory is that this drug mimics MBP and stimulates lymphocytes to have beneficial effects on injured spinal cords. Michal Schwartz and colleagues recently reported that copaxone is remarkably neuroprotective in the rat spinal cord contusion model. This drug is now recommended as the preferred treatment for relapsing-remitting form of multiple sclerosis. Proneuron recently signed a deal with Teva Pharmaceuticals to develop this drug for spinal cord injury. I expect this drug to go into clinical trial for human spinal cord injury relatively rapidly.
- Nogo vaccination.
3. Neurotrophic therapies (subacute and chronic) These therapies promote axonal growth. These include the classic growth factors (neurotrophins), as well as small molecules that appear to promote axonal sprouting. Note that many of these molecules also appear to block the axon's response to growth inhibitors.
- Protein growth factors. Cells secrete many proteins that stimulate growth of other cells. For the nervous system, the most studied class of growth factors are the neurotrophins.
- Neurotrophins (NGF, BDNF, NT-3, NT-4). These factors were discovered in the 1980's and are the most studied of neurotrophic factors, none of these factors are currently being developed for spinal cord injury. Bunge, et al. have suggested that combinations of these factors with other treatments such as matrigel bridges stimulate axons to grow when infused intrathecally to the spinal cord. NT-3 has also been reported to improve the regenerative effects of IN-1 (the antibody that blocks Nogo, the white matter growth inhibiting protein). However, some of the neurotrophins may have undesirable side-effects. Nerve growth factor or NGF, for example, has been reported to be associated with the development of neuropathic pain in animals. BDNF may suppress appetite when given directly to the brain. On the other hand, BDNF may have some beneficial effects on innervation of the bowel and reduce constipation. None of the these factors cross the blood brain barrier and needs to be given directly to the brain or spinal cord.
- Fibroblast growth factor**. This protein stimulates proliferation of stem cells. It comes in two forms, acidic and basic. The latter is used for stimulating stem cells while the former is part of the growth factor cocktail that Henreich Cheng uses, so from this perspective, it is in clinical trial.
- Glial derived neurotrophic factor (GDNF). This neurotrophic factor is secreted by glial cells. Like the other growth factors, GDNF is upregulated in the acutely injured spinal cord.
- Purine Nucleotides. Nucleotides are ubiquitous molecules that form the major building blocks for DNA and RNA. In addition, they form the backbone for critical intracellular messengers. Adenosine and guanosine are purine nucleotides that the the key components of ATP, ADP, cAMP, and GTP. Although adenosine receptors have long been known to be present on cells, the finding that the purine nucleotides have growth promoting effects came as a surprise. It appears that these effects of the purine nucleotides are not receptor mediated but directly involve some cytoplasmic or nuclear mechanism.
- AIT-082**. This is a modified guanosine derivative, sometimes called leteprinum potassium. It has been reported to stimulate proliferation of stem cells, to protect neurons, and also stimulate axonal growth in culture. It can be taken orally and some preliminary results suggest that the drug improves cognition in patients with Alzheimer's disease. Developed by Neotherapeutics, this drug is currently in phase 2 clinical trial for spinal cord injury at four centers: Ranchos Los Amigos in Downey CA, Craig Rehabilitation Center in Denver CO, Gaylord Rehabilitation Center in Connecticut, and the Jefferson Medical Center in Philadelphia PA. The current trial is restricted only to people within two weeks after injury.
- Inosine. This is a nucleotide that is the byproduct of adenosine metabolism. Larry Benowitz at Harvard University reported that inosine remarkably stimulated sprouting of the corticospinal tract in rats. Boston Life Science Inc. (BLSI) is developing this drug.
- AF-1. This is an axonal growth factor that was discovered by Larry Benowitz and is also being developed by BLSI. It has not yet been studied in spinal cord injury but it apparently stimulates axonal regeneration in the optic nerve.
- Adenosine. This is one of the most common nucleotides in the body, being a component of ATP, ADP, and AMP, as well as being one of the nucleotides that form the genetic code of DNA. Neurons have adenosine receptors. A recent report from Moses Chao and colleagues suggest that adenosine stimulates axonal growth in culture.
- Cell adhesion molecules. Cells express molecules on their surfaces that bind to other molecules. One particular family of cell adhesion molecules is of interest. Called the immunoglobulin (Ig) superfamily, these molecules have repeating Ig-like domains. These molecules not only adhere to each other but also serve as receptors that promote or inhibit growth. These molecules are evolutionarily conserved and analogs are present in all animals from worms to human, suggesting that they play a very important role in development and growth.
- L1-Fc or L1 fragments. L1 is a cell adhesion molecule that is expressed by growing axons. It is responsible for axons growth together in bundles. It is present in tissues that can regenerate, i.e. peripheral nerve, but is absent from the mature central nervous system. Two cells that promote axonal growth also strongly express L1: Schwann cells and olfactory ensheathing glia. L1-Fc is a soluble form of this cell adhesion molecule that can applied like a drug. Unfortunately, L1 is a very large molecule and difficult to manufacture and apply. Getting a soluble form of the molecule that promotes regeneration has turned out to be a more difficult task than first anticipated.
- Tenascin. This is another cell adhesion molecule that is present in the spinal cord. It may have some properties similar to L1.
- Laminin. This is a cell adhesion molecule that is prominently expressed in the peripheral nervous system and in collagen. Most biomaterials contain this molecule. Axons love to grow on this material.
- Hormones. Many hormones play a major role in cell metabolism, growth, and behavior. Chief amongst these are the pituitary hormones and the steroid hormones that they control. The pituitary hormones are usually relatively simple polypeptides while the steroid receptors are derived from cholesterol and other ring compounds.
- Adrenocorticotrophic hormone (ACTH). This is the pituitary hormone that stimulate the adrenal glands to make and release steroid hormones. It has long been reported to enhance peripheral axonal regeneration.
- Melanotropin (a part of ACTH). This molecule has been reported to be neuroprotective and possibly reparative. It is considered by some to be the active fragment of ACTH.
- Growth hormone. This pituitary hormone of course is responsible for the growth of many parts of the body. It is also involved in the body's response to glucose.
- Estrogen. Many researchers have reported that estrogen and related hormones can stimulate axonal growth and repair.
- Testosterone. Some researchers have reported the testosterone can enhance peripheral nerve regeneration.
4. Neurorestorative therapies. Many axons become demyelinated at the injury site. Although some remyelination occurs, many of the axons remain unmyelinated or are abnormally myelinated. Some therapies may be able to restore function to the spinal cord by increasing the excitability of axons so that they can conduct despite not being well-myelinated. Other therapies include serotonin to stimulate the central pattern generator and anti-spasticity therapies that counteract imbalance of neurotransmitters. The hallmark of such therapies is that their effects are temporary.
- Potassium channel blockers. Voltage-sensitive potassium channels play a major role in regulating the duration of action potentials (the signals that axons conduct). Normally, action potentials in axons are mediated by sodium channels at the nodes of Ranvier (the gaps of exposed axons between myelin segments) with little or no contribution from potassium channels. However, potassium channels are present on axons, usually underneath myelin. However, when axons are demyelinated, these channels become exposed and their activation causes not only current leakage but also shortening of action potential duration. Blockade of such channels can restore conduction to demyelinated axons.
- 4-aminopyridine**. This is a small molecule that can be taken orally and crosses the blood brain barrier. It blocks voltage sensitive potassium channels and apparently increases the excitability of demyelinated axons so that they can conduct action potentials. In addition, the drug increases the amount of neurotransmitter released per action potential. For people who have demyelinated axons crossing the injury site, this drug may improve motor and sensory function. The drug is being dispensed by compounding pharmacies in the United States, on prescription from a doctor. Acorda Therapeutics is developing a sustained release formulation that is currently in phase 3 clinical trial.
- 2, 4-diaminopyridine*. This is an analog of 4-aminopyridine that apparently does not cross the blood brain barrier as well but affects peripheral nerves. Thus, this drug has been in clinical trial for people with Guillain-Barre and other peripheral demyelinating conditions.
- Neurotransmitter therapies. Many neurotransmitters play a role in regulating neuronal excitability. Many of the neurotransmitters are amino acids. For example, glutamate activates a family of receptors including the NMDA and AMPA receptors, acetylcholine and adrenergic agonists activate their receptors, GABA activates inhibitory receptors GABA-A and GABA-B, and glycine also activates inhibitory receptors. These receptors often gate sodium, calcium, and chloride channels, as well as turn on various intracellular messengers. Thus, neurotransmitter receptor agonists and antagonists play a role in regulating the excitability of neurons and their growth. Several neurotransmitters have been reported to have potential regenerative effects.
- Serotonin. This is an important neurotransmitter that play an important role in modulating neuronal excitability, appetitie, inflammation, and vascular changes. Sometimes called by its chemical name 5-hydroxy-tryptamine or 5-HT, serotonin affects many different receptors and there are specific analogs that bind to particular serotonin receptors. For example, a drug called quipazine activates 5-HT2A receptors that are present on axons and increase the excitability of axons. This drug therefore may have effects similar to 4-aminopyridine.
- Clonidine*** is an alpha-2 adrenergic receptor agonist. Although the drug is used to reduce blood pressure, it also reduces spasticity. When administered intrathecally, clonidine stimulates locomotion by activating the central pattern generator for locomotion in the lower spinal cord. It is frequently used to activate locomotion in spinalized (transected) animals in laboratory studies. About 20 years ago, Eric Natchi reported that clonidine improves recovery in animals and patients with spinal cord injury. Although this work could not be confirmed, the possibility that clonidine may have effects on axonal growth has not yet been ruled out.
- Tizanidine*** is a centrally acting alpha-2 adrenergic receptor agonist that was approved by the FDA for treatment of spasticity. The drug has effects similar to clonidine but because it is supposed to have more central effects, some of the side-effects profile is different.
- Electrical stimulation. Electrical stimulation activate neurons and muscles. Activity in turn changes the metabolism and growth behavior of cells. For example, muscles and cells that are inactive tend to undergo atrophy. Electrical stimulation is an important means of preventing and reversing atrophy.
- Stimulation of the central pattern generator**. Several groups have now reported successful stimulation of the central pattern generator (CPG) in the spinal cord, producing locomotor activity. This is of interest because people who are barely house-hold walkers can walk much more efficiently and effectively with the stimulator. Apparently the stimulator can activate patterns of walking muscles that the persons cannot activate voluntarily. It is likely that this stimulation approach will make reversal of learned non-use much easier and faster to accomplish than by the current approach of forced use weight-supported treadmill ambulatory training.
- Functional electrical stimulation***. Electrical activation of muscles and nerves is called functional electrical stimulation (FES). Electrodes applied to muscles, particularly from the skin surface, activate only the surface muscles whereas activation of nerves generall will produce deeper and greater muscle responses. Stimulation of the nerves is sometimes called functional neurostimulation (FNS). Hunter Peckham, Byron Marsolais, and their colleagues at Case Western University developed a several sophisticated FES systems to control muscles in the hand and legs. One has been commercialized. Called the Freehand system, impanted electrodes in the hands are controlled by a computer-driven stimulator. Although FES and FNS are not generally considered a restorative therapy, it is very likely to play a major role in the restoration of motor function in clinical trials. Muscle activation stimulates muscle growth while inactivity causes rapid atrophy of muscles. FES can reverse atrophy even of muscles that have been denervated. In addition to providing exercise that prevents atrophy, FES and FNS can restore useful function to the arms, hands, and legs. A thriving industry has grown up around this concept and over 100 companies now offer a variety of commercial stimulators for exercise and functional purposes.
- Forced-use training. Other approaches can be used to stimulate activity
- Constraint-induced therapy.** In 1999, Edward Taub reported that a new rehabilitation approach called "constraint-induced therapy" can produce large improvements in motor function of limbs that have been paralyzed for many years. The signature therapy involves constraining movement of the less-affected structures and thereby forcing subjects to use the impaired limb. This results in a massive use-dependent cortical reorganization. Similar methods are now being applied to restoring function in patients who are hemiplegic from stroke or spinal cord injury. The theory underlying this therapy is that non-use results in a loss and reorganization of both cortical and spinal circuits.
- Supported treadmill ambulation**. Forcing the patients to use the impaired limbs can restore these circuits by constraining the unimpaired limb is only useful in hemiplegics. In 1995, Wernig, et al. at the University of Bonn reported a stunning results. They trained 89 incompletely paralyzed para- and tetraplegics to walk with partial weight supported treadmill walking, compared to 64 spinal-injured subjects that were treated conventionally. This training was called laufband (LB) therapy. Of the trained patients, 33 were 0.5-18 years after injury, wheelchair bound, and unable to stand or walk without help by others. By the end of 3-20 weeks of training, 76% of these were walking independently. Patients who were able to walk before LB training were improved by the training. For example, of 44 patients with chronic spinal cord injury (>6 months), 6 were capable to staircase climbing before training and 34 were capable of doing so after training. In a followup study in 1998, Wernig, et al. reported that 31 of 35 patients continued to be able to locomote independently. These results prompted the National Institutes of Health to initiate a multicenter study of partially supported ambulatory training in people with spinal cord injury in the United States. Some preliminary reports from individual centers suggest that treadmill training does restore locomotor functioning in many patients with incomplete spinal cord injury. These findings are important and worrisome because it suggests that regenerative therapies, if they are successful, may not result in apparent locomotor improvement unless the treatments are combined with locomotor training.
- Biofeedback therapy***. Biofeedback therapy is another form of training that "forces" an individual to use particular muscle groups. Biofeedback training has been practiced in the United States for many years. Most major rehabilitation centers have some form of biofeedback training facility. For example, the University of Miami Biofeedback Laboratory, headed by Dr. Bernie Brucker, has long offered biofeedback therapy for people with spinal cord injury. The theory underlying biofeedback training is as follows. Electromyographic (EMG) recording of muscle activity allows users to perceive and quantify muscle activity. On any given day, subjects will have a range of voluntarily activatable muscle activity. By focusing attention on a particular muscle, subjects usually can enhance their voluntary activation of EMG activaty so that it is more consistently at the higher level of activation. When subject reach that criterion, they are then shifted step-wise to higher and higher criterion. It is important to note that such training, while effective in promoting better voluntary activation of muscles, does not necessarily restore coordination nor is the training generalizable to other muscles that are not directly trained. Nevertheless, such training may be a useful adjunct to other "forced-use" training paradigms to improve functional manifestations of regenerative treatments in the future.
5. Neuroregenerative therapies. Regeneration implies long distance growth of axons. Neurotrophic factors may cause axons to grow but other therapies are necessary to get the axons to grow all the way home. Three major classes of molecules have been reported to inhibit axonal growth in the spinal cord. These include Nogo, MAG (myelin-associated glycoprotein), and CSPG (chondroitin-6-sulfate proteoglycans). Some of these therapies are antibodies that bind inhibitors (IN-1, anti-Nogo antibodies, therapeutic vaccine to stimulate antibodies) or break down inhibitors (chondroitnases). Others target the receptors that respond to the inhibitors or their upstream and downstream promoters.
- Nogo blockers. Nogo is a protein that was discovered by Martin Schwab to inhibit axonal growth. Present on white matter (WM), Nogo is believed to the one of the most important inhibitors of axonal growth in the spinal cord. An antibody called IN-1 binds Nogo and was the first treatment found to regenerate the spinal cord of adult mammals. Unforunately, the first IN-1 antibody was a mouse antibody of the IgM kappa class. Because IN-1 is a mouse antibody, it of course cannot be given to humans. The molecule not only would be rejected immediately but would run a high risk of producing a severe allergic response in people. Furthermore, IgM kappa are huge antibodies (Megadalton) that are difficult to manufacture. Although many companies now have experience making IgG antibodies, few companies have any experience successfully manufacturing large quantities of an IgM antibody and taking it to clinical trial. Furthermore, Schwab, et al. was not able to show that IN-1 blocks Nogo when given directly to rats. In virtually all their experiments, they implanted the hybridoma (the lymphocyte tumor that make the IN-1 antibody) into the brain so that the cells would make the IN-1. However, they did report results using IN-1 to block myelin-growth inhibition in vitro. Thus, it is likely that Schwab, et al. could not produce a sufficient working amounts of IN-1.
- Humanized IN-1. This is a humanized form of IN-1. Created through recombinant molecular techniques, this antibody contains the recognition end of the mouse IN-1 attached to the constant regions of human antibodies. Schwab, et al. reported in 2000 that this antibody improved regeneration and locomotor recovery in rats with overhemisections of the spinal cord. This is the antibody that Novartis is developing for clinical trial. Novartis is currently testing the antibody in monkeys in preparation for phase 1 human clinical trials.
- Human anti-Nogo antibodies. In January 2000, four groups simultaneously reported the isolation of the gene for Nogo. The identification of the gene allowed production of the Nogo protein for the first time. Once the protein is available, it can be used to inoculate animals to obtain antibodies against Nogo. Several such antibodies have now been reported. Another possibility is to use Nogo as a vaccine to induce the body to form its own antibodies against Nogo. Note that actual human antibodies or if such a Nogo vaccination approach is developed and shown to be effective, IN-1 itself may never proceed to clinical trial because IgG human antibodies or a Nogo vaccine will be easier to develop and manufacture.
- Nogo receptor blockers
- Nogo fragments. Stephen Strittmatter from Yale University recently reported the discovery of the Nogo receptor. He further found that certain fragments of Nogo can block the Nogo receptor. In the recent Society for Neuroscience meeting, he reported that these Nogo fragments stimulate regeneration in transected rat spinal cords. The Nogo fragments are now being developed by Biogen, a major company with substantial resources. The Nogo fragments, although smaller than Nogo itself, are nevertheless two large to cross the blood brain barrier. It will have to be given intrathecally. In theory, blockade of the receptor should be more effective than using an antibody (IN-1) to cover up the Nog. The fragments will also be easier to manufacture and, since they are human Nogo, they should not evoke any immune response or inflammation.
- Chondroitinase ABC (CABC). This is a bacterial enzyme that breaks down chondroitin-6-sulfate proteoglycans (CSPG) that have been reported to stop axonal growth. Jerry Silver and his colleagues have long reported by CSPG stops axonal growth in the brain and spinal cord. CSPG is also the extracellular matrix protein that repels migrating cells and serves to direct growth. CABC has been used for treating extruded spinal discs which has chondroitin. Several recent studies for James Fawcett's laboratory have reported that chondroitinase ABC facilitate regeneration in the brain and spinal cord. Being a bacterial enzyme, CABC may stimulate inflammation and break down extracellular matrix proteins in the spinal cord. To date, the studies reporting CABC in spinal cord are still preliminary and involve application of CABC to the spinal cord after discreet lesions of the corticospinal tract. It has not yet been tested in any chronic spinal cord injury and not yet in contusion models that are more like most human spinal cord injury.
- Therapeutic vaccines
- Spinal cord homogenate. Sam David and colleagues reported in 1999 that vaccinating mice with spinal cord homogenates can remarkably promote spinal cord regeneration and locomotor recovery. They recently showed that serum obtained from vaccinated mice and transferred to other mice will enhance recovery, suggesting that the vaccination is working by inducing antibodies. More recently, Ousman & David (2001) reported the myelin inoculations have similar effects but may be acting through cellular immunity mechanisms.
- Myelin proteins. Hauben, et al (2001) from Michal Schwartz's laboratory reported in 2001 that vaccinating rats with myelin basic protein activated t-lymphocytes and these lymphocytes protect the spinal cord and may stimulate regeneration. Hauben, et al. (2001) recently reported that inoculating rats with Nogo likewise will stimulate repair and perhaps regeneration in rats after spinal cord injury. Schwartz, et al. have proposed that myelin proteins elicit a family of cellular mediated immune responses that are neuroprotective. They argue that the cellular response is faster by directly activating lymphocytes that then migrate to the injury site.
- Glatiramer acetate (Copaxone). In November 2001, Schwartz and colleagues reported that this drug also activates lymphocytes and have remarkable neuroprotective and possibility reparative effects on the spinal cord. This drug, also called co-polymer 1, is a mixture of polymers consisting of four amino acids that apparently mimic parts of a molecule called myelin-basic protein (MBP) that is one of the proteins in myelin, as the name implies. As pointed out above, Schwartz, et al. showed that MBP inoculations are neuroprotective and may even stimulate axonal regeneration. Thus, glatiramer acetate may work like MBP in stimulating the immune system, activating lymphocytes to exert beneficial effects on the spinal. Note the glatiramer acetate is currently the recommended first-line therapy for the relapsing-remitting form of multiple sclerosis where it has the more benefit and less side effects than betaseron (inteferon-beta) and other interferon-like drugs. Thus, its safety profile is well established. If further animal studies support the beneficial effects of glatiramer acetate in animals, this drug can and should go rapidly into phase 2 clinical trials.
- Upstream messenger promoters. "Upstream" refers to messengers that are close to the receptor that is activated by an instigating factor. For example, an upstream messenger that tells a neuron to start growing is cAMP (cyclic adenosine monophosphate). Drugs that increase cAMP levels in the neurons tend to stimulate neuronal growth. Note that cAMP is a general messenger that mediate many other responses of cells to stimuli and therefore general increases of cAMP may have multiple side-effects.
- Rollipram** . Marie Filbin and colleagues reported that neurons that combinations of neurotrophins stimulate cultured neurons to grow in the presence of myelin growth inhibitors such as Nogo and MAG. They found that the neurotrophins increased cAMP, a common intracellular messenger. When they applied cAMP to the neurons, they grew and ignored all the myelin growth inhibitors. They also found that they could apply cAMP analogs to the dorsal root ganglion neurons (the sensory neurons) and get them to regenerate in the spinal cord. So, they proposed using Rollipram. This is a long-established drug that blocks phosphodiesterase-4 and has been used as an antidepressant. Rollipram selectively blocks phosphodiesterase in the central nervous system. Rollipram is currently undergoing clinical trial in multiple sclerosis where it has been hypothesized to promote remyelination. injury. Several laboratories are now testing rollipram in rat spinal cord injury models. If and as soon as these studies show positive results, this is a drug that will go rapidly into clinical trial. One of the most exciting possibility is using Rollipram in conjunction with Schwann cell or other transplants that can provide a bridge for axonal growth across the injury site. The Rollipram can help the axons get from the bridge back into the spinal cord. Marie Filbin received the 2000 Ameritec award for this work.
- Theophylline** Theophylline is an adenosine receptor antagonist. Long used to treat asthma, theophylline has a well known side-effect profile and safety record. Harry Goshgarian at discovered several years ago that theophylline stimulates plasticity of the phrenic nucleus and axonal growth. The phrenic nucleus innervate the diaphragm. Recently, Nantwi & Goshgarian (2001) compared theophylline with other alkyxanthines and showed that those that did not penetrate the blood brain barrier were ineffective and that theophylline was the most potent of the akylxanthines tried. Goshgarian has been working with physicians giving theophylline to ventilator dependent patients to see if they can be weaned. Although there have not been any formal studies of theophylline in spinal cord injury models, much is known about the effects of theophylline on the central nervous system. This is the kind of therapy that probably should go to clinical trial directly. The risks of therapy would be very low.
- Alternating electrical currents** Richard Borgen at Purdue University has long reported that electrical currents stimulate axonal growth in the spinal cord. One of the problems is that the axons tended to grow towards one electrical pole and it was unclear how to stimulate both motor and sensory axons to grow down and up te spinal cord. Recently, Borge reported that alternating currents will stimulate growth in both directions in dogs. Last year, Purdue University initiated a clinical trial of AC electrical stimulation to stimulate repair and regeneration in humans. Restricted to people who are within two weeks after injury, the trial proposes the implantation of an electrical alternating current stimulator. Another clinical trial is being carried out with electrical currents in Dublin, Ireland. The use of electrical stimulation for stimulating axonal regeneration was given a boost lately. Ming, et al. (2001), including contributions from Moo Ming Poo and Marc Tessier Lavigne, recently found that alternating electrical currents increase cAMP levels in neurons. This finding helps explain decades of reports by other neurophysiologists that electrical currents modify axonal growth.
- GM1 (monosialic ganglioside, Sygen)* . Monosialic ganglioside or GM1 has long been known to stimulate neuronal growth in cell cultures. It is called a glycolipid because sialic acid is a carbohydrate (glyco) and monosialic indicates that there is only one sialic acid on the molecule, and the 1 indicates that it is in the first position. Ganglioside is a lipid. GM1 is naturally present in fairly high concentrations in brain and other organs with membranes. It is very interesting that GM1 is the site that cholera toxin binds to on cells. Cholera toxin strongly stimulates adenyl cyclase which in turn produces cAMP. However, when GM1 is used as a therapy, it is not clear how it might be working from the extracellular space. Presumably, GM1 is taken up by the cells and incorporated into the membrane of the cells. Many investigators have reported the GM1 applied to the cells will prevent translocation of protein kinase C (a calcium-sensitive membrane-bound enzyme that plays a crucial role in phosphorylating and activating other enzymes in the cell). At least one protein kinase C, i.e. PKC-gamma, has been implicated to play a role in neuropathic pain, one of the reasons why GM1 has been used extensively to suppress pain in patients with peripheral neuropathy. In 1991, Fred Geisler published a paper in the New England Journal of Medicine reporting that 100 mg of GM1 given intramuscularly daily for 6 weeks starting 48 hours after spinal cord injury resulted in better locomotor recovery in half of 37 patients. Subsequently, Fidia Pharmaceuticals, the company that makes GM1 funded a multicenter clinical trial in the United States. That trial of about 800 patients showed that GM1 accelerated recovery of both motor and sensory function up to 6 weeks but over the following 3-4 months, the placebo-control group caught up with the GM1-treated group so that the groups did not differ significantly from each other. That study was just published in Spine, described in the Research section, and it can be downloaded in .pdf format from this site. Fidia unfortunately is no longer in a financial position to fund a further trial and it is likely that the FDA will require a second pivotal trial to demonstrate efficacy (since this trial did not meet the criterion for significant number of GM-1 patients recovering 2 or more levels on the Benzel functional scale.
- Upstream messenger blockers. Other upstream messengers contribute to the response of neurons when they encounter extracellular growth inhibitors, such as Nogo, MAG, and CSPG. Two such messengers are called Rac and Rho had been earlier reported to be the intracellular messengers mediating the response of neurons to extracellular matrix proteins called semaphorins. Rho is a GTPase (a enzyme that breaks down guanosine triphosphate) and apparently mediates the inhibitory responses of the cell to growth inhibitors. Thus, blockade of Rho should stimulate regeneration and cause neurons to ignore growth inhibitors.
- Rho blockers: C3. Lisa McKerracher and colleagues at the Montreal Institute of Neurology used a bacterial toxin called C3 block Rho. Made by the bacterium Clostridium botulinum, C3 is also called botulinum ADP-ribysyltransferase C3. An exo-enzymes that specifically inhibit Rho GTPases, C3 remarkably stimulating axons to grow on tissue culture that contained several growth inhibitors including Nogo, MAG, and CSPG. Recently, McKerracher, et al. reported at the Society for Neuroscience meeting in November 2001 (San Diego) that intrathecal application of C3 stimulates regeneration and functional recovery in rats after spinal cord contusion injuries. Rho is a GTPase (an enzyme that breaks down guanosine triphosphate) and is activated by Rho kinase (an enzyme which phosphorylates Rho). A number of Rho kinase inhibitors are also known. For example, an experimental drug Y-27632 blocks Rho kinase.
- Recombinant C3 that crosses blood brain barrier. C3 does not pass the blood brain barrier. However, McKerracher, et al. started a company called BioAxone to develop a novel recombinant form of C3 that can pass through the blood brain barrier.
- Downstream growth messengers
- Spermidine. Marie Filbin has been looking for the downstream messengers responsible for axonal growth. She recently reported evidence that these messengers appear to be spermidine, a polyamine that comes from ornithine-->putrescine-->spermidine-->spermine. The polyamines have long been reported by other investigators to have a variety of effects on axonal growth. It may be possible to apply spermidine or its precursors directly or increase the cellular concentration of spermidine by using drugs to inhibit enzymes responsible for breaking down spermidine.
- Other polyamines. Application of ornithine and putrescine is another way of increasing spermidine in cells.
6. Neuroconstructive. Injury damages cells in the spinal cord. The injury site becomes a wasteland that may not be conducive to axonal growth. Also, injury kills neurons and oligodendroglial cells die. Stem cells provide a promising approach to replacing these cells. Stem cells are pluripotent cells that produce multiple types of cells, including cells that remyelinate spinal axons, that improve the environment of the spinal cord for axonal regeneration, and replace neurons that have been lost. These cells come from many different sources, from animals, humans, embryonic, fetal, adult, brain, and other tissues. In addition to stem cells, many groups are also assessing precursor cells and differentiated cells. These cells produce only a limited type of cells that may have specific functions or effects in the spinal cord. Some of these cells, i.e. the olfactory ensheathing glia, migrate rapidly in the spinal cord and appear to facilitate regeneration. Others promote myelination and provide bridging materials. The best timing and sits of cell transplants, as well as types of cells, are not well understood.
- Fetal stem cell grafts
- Porcine neural stem cells xenografts**. Diacrin developed a proprietary method of blocking rejection of pig cells, using antibodies against surface MHC class I antigens. Although this method does not prevent rejection of solid organs, it apparently allows non-rejection of porcine fetal cell suspensions transplanted to the brain and spinal cord. Over 100 patients have received various forms of porcine xenografts for stroke, Parkinson's disease, and Huntington's disease. A clinical trial of porcine fetal stem cells has begun at Washington University in St. Louis and Albany Medical Center in New York. A total of six patients with chronic spinal cord injury will have received porcine cell transplants in this trial. There is substantial concern about the dangers of pig viruses transmitting to human cells. Apparently, the strain of pigs used by Diacrin possess viral genomes that do transfer to human cells when the porcine cells are co-cultured with human cells, particularly germ cells such as sperm. For that reason, the FDA required all subjects of these trials to agree not to have children, to be registered for lifetime with the FDA, and to allow their bodies to be autopsied in case of death.
- Human fetal neural stem cells*. Stem cells can be isolated from aborted human fetuses. Several center have already begun clinical trials of such cells in Russia and in China. For example, Samuil Rabinovich in Novosibirsk and A. F. Bruhovetsky in Moscow apparently have each transplanted fetal stem cells into the spinal cord of as many as 15 patients with chronic spinal cord injury and preliminary reports suggest that functional improvements in the patients but the studies have not yet been peer-reviewed. Such fetal stem cell transplants are also apparently being carried out in China as well.
- Adult stem cell autografts
- Human bone marrow stem cells. The bone marrow contains a population of cells called mesenchymal stem cells Sasaki, et al. (2001) recently reported the bone marrow cells rapidly remyelinated spinal cords of rats demyelinated by x-irradiation.
- Human fat stem cells
- Human adult nasal epithelial stem cells
- Embryonic stem cell isografts and heterografts
- Human embryonic stem cells
- Human cloned embryonic stem cells
- Enteric glial stem cells
- Adult isografts (from appendix)
- Adult heterografts (from cell lines)
- Omentum transpositions?
- Olfactory ensheathing glia
- Human OEG autograft
- Human fetal OEG heterografts*
- Porcine fetal OEG xenografts.
- Schwann cells
- Schwann cell autografts* (from one's own peripheral nerve)
- Heterografts (cell lines)
- Precursor cells
- Neuron-restricted precursor cells (NRPs)
- Glial-restricted precursor cells (GRPs)
- Oligodendroglial precursor cells (O2A)
7. Neurogenetic. Many genes are associated with regeneration, pain, or remyelination. It is now possible to insert genes into the spinal cord, either indirectly (ex vivo gene therapy) by inserting the genes into cells that are then transplanted to the cord or directly (in vivo gene therapy) by inserting the genes directly into spinal cord cells. Until recently, scientists used viruses to insert genes but there are new methods that allow the insertion of gene directly or indirectly into cells without viruses. Please note that as specific genes are identified, this section will identify specific genetic factors.
- Genetically modified cell transplant
- Neurotrophin secreting fibroblast (Tuszynski's treatment)
- Sertoli cell transplants (anti-immunogenic cells from sperm ducts)
- DNA insertion (gene gun, liposomal vectors)
- Neurotrophin genes
- Cell adhesion molecules (i.e. L1)
- Viral vectors
- Adenoviral vectors
- Retroviral vectors
©Wise Young PhD, MD