Patent Application: US-71621807-A

Abstract:
the invention provides a method of reducing spasticity in a mammal involving administering modafinil or related compounds . the spasticity can be secondary to multiple sclerosis , spinal cord injury , stroke , brain injury , and other causes .

Description:
the term “ reduce hyper - reflexia ” as used herein refers to reducing the amplitude of the h - reflex measured at 1 , 5 , or 10 hz as normalized to amplitude measured at 0 . 2 hz stimulation . in other words , it refers to restoring low frequency - dependent depression of the h - reflex to closer to control levels . more preferably the h - reflex amplitude is reduced at 5 or 10 hz , most preferably at 5 hz . the invention involves the discovery that administration of modafinil decreases ( normalizes ) h - reflex amplitude in spinally transected rats . humans having sci develop spasticity , not immediately after the injury , but over a course of several weeks after the injury . spinally transected rats are an animal model for sci in humans . a major component of spasticity is exaggerated reflexes or hyper - reflexia . the hoffman reflex or h - reflex is a standard measure used by investigators to quantify hyper - reflexia . it is the electrical analogue of the classic tendon jerk reflex . it is a compound electromyographic response elicited by the synaptic activation of motoneurons by muscle afferents following stimulation of muscle nerves . it can be measured as described in section 1 of the example below . the h - reflex amplitude is a standard quantitative measurement that has been found to correspond to hyper - reflexia seen in spasticity ( 28 , 40 , 41 ). the example below shows that passive exercise with a motorized bicycle reduces h - reflex amplitude , and that this effect increases as the time period of daily one - hour exercise increases from 15 days to 90 days . the same effect of h - reflex amplitude decrease is seen with passive exercise in a single human sci patient ( section 2 of the example below ). thirty days of daily modafinil administration to spinally transected rats without any passive exercise decreased ( normalized ) the h - reflex amplitude to the same extent as 30 days of exercise ( section 4 of the example ). the most commonly used drug to treat spasticity , baclofen , is a gabab receptor agonist and has the side effect of causing drowsiness . modafinil in contrast does not cause drowsiness , and in fact is used to treat narcolepsy , but its mechanism of action is unknown . thus , it appears to act by a different mechanism . in this regard , it is interesting that passive exercise , which also reduces spasticity , increases expression of the nerve gap junction protein connexin 36 , which would increase gap junctions and increase electrical communication between neurons . other compounds that are structurally similar to modafinil and induce similar effects can also be used in the present methods to treat spasticity . these include compounds of formula i : wherein each of rings a and b may optionally be substituted by one or more of the groups f , cl , br , cf 3 , no 2 , nh 2 , c 1 - 4 alkyl , c 1 - 4 alkoxy , and methylenedioxy ; a is hydrogen ; c 1 - 4 alkyl ; c 1 - 4 hydroxyalkyl ; a group of the formula — y — nr 2 , wherein y is a divalent linear or branched chain c 1 - 4 hydrocarbon radical and each r is independently h or c 1 - 4 alkyl ; or a hetercyclic group that has five to seven ring members , including one or two heteroatoms selected from n and o , and optionally substituted with 1 or 2 c 1 - 4 alkyl ; this family of modafinil relatives is described in u . s . pat . no . 4 , 177 , 290 . in particular embodiments of the compounds of formula i , z is & gt ; ch — so —. in preferred embodiments , of the compound of formula i , a is hydrogen , hydroxymethyl or β - morpholinoethyl . in particular embodiments of the methods of the invention the spasticity is secondary to ( i . e ., is a consequence of ) multiple sclerosis , a spinal cord injury , a stroke , or a brain injury . in a preferred embodiment , the spasticity is secondary to a spinal cord injury . in particular embodiments of the invention , the spasticity is secondary to a neurological injury ( i . e ., an event that harms neurological tissue , including a spinal cord injury , brain trauma , and a stroke ). where the spasticity is secondary to a neurological injury , in particular embodiments , a compound of formula i is administered after spasticity has set in , and in other embodiments before spasticity has had a chance to set in . that is , modafinil may be used to normalize excessive reflexes , a major element of spasticity , once they have manifested , as well as to prevent the onset of excessive reflexes after injury or disease but before hyper - reflexia and its attending spasticity has become manifested . thus , where the spasticity is secondary to a neurological injury , in particular embodiments a compound of formula i is administered initially within 16 weeks of the injury , preferably within 4 weeks of the injury , more preferably - within 1 week of the injury before spasticity fully develops in order to prevent development of spasticity . in other embodiments , the modafinil or other agent of formula i is administered initially after spasticity is developed , e . g ., after about 16 weeks after the injury . the invention is not limited to a particular mechanism of action . but in section 4 of the example below it is hypothesized that spasticity and hyper - reflexia develop over a period of weeks after spinal cord injury because of a decrease in expression of gap junction proteins and a resultant decrease in gap junctions in spinal neurons below the injury , and thereby a decrease in electrical coupling of these neurons . connexin 36 is the primary gap junction protein in neurons . section 4 of the example below shows that connexin 36 gene expression is decreased after spinal cord injury in rats in spinal neurons below the site of the injury . passive exercise is shown to reverse this decrease of connexin 36 expression . it is hypothesized that modafinil is effective because it increases connexin 36 gene expression and thereby increases electrical coupling of neurons . the invention will now be illustrated by the following non - limiting example . restoration of frequency - dependent depression of the h - reflex by passive exercise in spinal rats spinal cord injury ( sci ) results in numerous deficits of the motor and sensory systems , including paralysis , anesthesia , and hyper - reflexia below the level of the lesion . hyper - reflexia is evident in both humans and non - human animals following sci and is a major component of spasticity . the physiological changes that have been postulated to contribute to hyper - reflexia include alpha motoneuron hyperexcitability ( 1 , 2 , 3 ), changes in the intrinsic properties of alpha motoneurons ( 4 , 5 , 6 , 7 ), reduced post - activation depression of transmission from ia fibers ( 8 , 9 ), synapse growth ( 10 ), alterations in morphology of alpha motoneurons ( 11 ), and decreased presynaptic inhibition of ia terminals ( 9 , 12 , 13 , 14 , 15 ). the time course of spinal changes after injury has been proposed to include an early postsynaptic mechanism , possibly involving an increase in excitability and / or receptor upregulation , and a late change involving presynaptic mechanisms possibly involving synaptic growth in spared descending pathways and in reflex pathways ( 10 ). one measure used by numerous investigators to quantify hyper - reflexia is the electrical analogue of the classic tendon jerk reflex , referred to as the hoffman or h - reflex ( 2 , 15 , 16 , 17 , 18 , 19 ). the h - reflex is a compound electromyographic ( emg ) response elicited by the synaptic activation of motoneurons by muscle afferents following stimulation of muscle nerves . thompson et al . investigated four measures of h - reflex excitability in a contusion model of sci in the rat ( 20 ). results of their studies led these researchers to conclude that rate - sensitive depression of the h - reflex was of particular importance in the assessment of hyper - reflexia following sci . other groups have reached similar conclusions regarding the importance of changes in h - reflex rate - sensitive depression as a measure of the effects of sci ( 21 ). in spinally intact individuals , the h - reflex demonstrates depressed amplitude , due to marked frequency - dependent depression , once stimulus frequencies reach or exceed 1 hz ( 22 , 23 ). however , frequency - dependent depression of the h - reflex is less evident in patients or animals with chronic sci ( 12 , 13 , 22 , 24 , 25 ). the present section of this example reports on a study to determine the effect of passive exercise therapy to restore frequency - dependent depression of the h - reflex in adult rats with complete spinal cord transections . a motorized bicycle was used to provide the passive exercise ( motorized bicycle exercise training ( mbet )). adult female sprague - dawley rats ( harlan , 200 - 250 g , n = 40 ) underwent a lower thoracic laminectomy under ketamine ( 60 mg / kg , i . m .) and xylazine ( 10 mg / kg , i . m .) anesthesia . a complete transection ( tx ) of the spinal cord was made by aspiration and the transected ends of the cord retracted , producing a 2 - 3 mm cavity . gelfoam was inserted into the cavity to facilitate hemostasis and the dura was closed over the tx site . muscle and skin were sutured in separate layers , and animals were provided with dextrose - saline ( 5 %, 1 ml / 100 g body weight , s . c .) to replace fluid lost during the surgical procedure . penicillin ( 5000 u , i . m .) was administered immediately postoperatively , and animals were transferred to an incubator maintained at 37 . 5 ° c . until fully recovered from the anesthetic . the urinary bladder of each animal was expressed manually twice daily until reflexive voiding was established ( 10 - 14 days ). animals were monitored for signs of urinary tract infection , and treatment with baytril ( enroflaxin 0 . 2 mg / day , i . m . for 10 days ) was instituted as needed . all procedures were approved by the institutional animal use and care committee at uams . one group of rats ( tx only 90d , n = 4 ) underwent no further treatment until reflex testing was carried out 90 days after tx , while a second group of transected animals ( tx only 30d , n = 8 ) underwent no training but was tested for h - reflex frequency - dependent depression after 30 days , and a group of intact rats served as nontransected controls ( control , n = 5 ). the remaining rats ( n = 23 ) were divided into five groups that received mbet daily . exercise was provided for either 15 days ( tx + ex 15d , n = 5 ), 30 days ( tx + ex 30d , n = 4 ), 45 days ( tx + ex 45d , n = 5 ), 60 days ( tx + ex 60d , n = 5 ), or 90 days ( tx + ex 90d , n = 4 ). the exercise regimen mbet involved suspending the rats on a sling with the hindlimbs hanging down and the hind paws strapped to the pedals of a bicycle - type device , which was driven by a motor . the pedaling motion flexed one hindlimb and simultaneously extended the contralateral one , while avoiding overstretching of either limb . cycling speed was 30 rpm . exercise sessions consisted of two 30 - min episodes with 10 min of rest in between . after the end of the training period , reflex testing was performed . the two tx only groups also underwent reflex testing 30 days or 90 days after tx , along with a group of intact rats ( ctl ), for comparison with the mbet experimental groups . animals were anesthetized with ketamine ( 60 mg / kg , i . m .) and maintained with 10 % doses as needed such that vibrissal and pinna pinch reflexes were absent . core body temperature was maintained at 36 ° c . using a thermostatically controlled heat lamp . a bipolar cuff electrode was placed on the tibial nerve for stimulation ( 0 . 1 ms pulses , cathode proximal on nerve ). exposed tissue was covered with mineral oil to prevent drying . a wire electrode was inserted subcutaneously in the digital interosseous muscles between the fourth and fifth metatarsals for emg recording as previously demonstrated ( 26 , 27 ), and referenced to a clip applied to the skin on the digits . a ground electrode was attached to the skin of the tail . recordings were made using amplifier ( grass p511 ) filter settings of 3 hz to 3 khz with the 60 hz notch filter in use . responses to the stimulus were digitized and averaged using a gw instruments ( somerville , mass ., usa ) digitizer module and superscoper software . stimulation of the tibial nerve under the calcaneal tendon produced two responses , an early m - wave ( 2 ms latency ), produced by direct activation of motoneuronal axons in the tibial nerve , and a later h - reflex ( 8 ms latency ), produced by activation of muscle afferents in the tibial nerve , which synapse monosynaptically on plantar motoneurons . the degree of stimulation that induced frequency - dependent depression of the h - reflex was determined . the reflex was first tested at 0 . 2 hz to determine threshold and maximal response levels . after discarding the first five responses in order to obtain an average of the stabilized reflex , averages of 10 responses were obtained . averages were compiled following stimulation at 0 . 2 , 1 , 5 , and 10 hz . the change in the response at various frequencies was calculated as the percent of the response at 0 . 2 hz in order to determine depression of the h - reflex as a function of stimulation frequency . following the frequency series testing , the h - reflex amplitude was confirmed at 0 . 2 hz for consistency . if the amplitude at recheck was less than 90 % of the initial amplitude , the data was discarded . at the end of the experiment , animals were euthanized with an overdose of barbiturate ( nembutal ) and the tx was confirmed either visually or histologically following transcardial perfusion with paraformaldehyde ( 4 %) and sucrose ( 20 %). sensory testing or assessment of spasticity was not carried out in this series of animals . the amplitude of the h - wave was measured from the base line before the h - wave to the peak of the first ( and largest ) of its two components ( fig1 ). measurements from peak to peak of the two components gave similar results so that only the baseline to peak measures are reported here . results from animal groups were compared statistically using a two - way anova test . significant differences between groups were tested using the scheffe test , a conservative post hoc comparison . statistical significance was considered to be present at p & lt ; 0 . 05 . fig1 panel a , shows representative recordings from an intact control animal stimulated at 0 . 2 hz ( the 100 % response ), along with ( fig1 , panel b ) the response at 10 hz ( group mean ± se of the mean , ˜ 11 ± 4 % of the response at 0 . 2 hz ), and the response from a tx only 60d rat at 0 . 2 hz ( 100 %) ( fig1 , panel c ), and at 10 hz for comparison ( 68 ± 6 %) ( fig1 , panel d ). panels e and f of fig1 show the responses from a tx + ex 60d rat at 0 . 2 hz ( 100 %) and 10 hz ( 22 ± 7 %), respectively . these recordings demonstrate the main effects of higher frequency stimulation leading to marked frequency - dependent depression in intact animals , of tx leading to decreased ( increased percent ) frequency - dependent depression , and of mbet leading to a restoration of frequency - dependent depression of the h - reflex . fig2 is a graph of the habituation of the h - reflex following stimulation at 0 . 2 , 1 , 5 , and 10 hz in the following groups of animals : tx only 90d , tx + ex 15d , tx + ex 30d , tx + ex 45d , tx + ex 60d , tx + ex 90d , and control . the tx only 30d ) group was not included because of its similarity with the other longer duration unexercised transected group ( tx only 90d ). statistically significant differences between the tx only 90d group and the other groups at each frequency are denoted by a single (* p & lt ; 0 . 05 ) or a double (** p & lt ; 0 . 01 ) asterisk . anova of these groups showed statistically significant differences across stimulation frequency ( df = 3 , f = 115 . 11 , p & lt ; 0 . 001 ), across experimental groups ( df = 7 , f = 6 . 77 , p & lt ; 0 . 001 ), and interaction between frequency and groups ( df = 21 , f = 2 . 17 , p & lt ; 0 . 008 ). post hoc comparisons between all groups were undertaken , and those against the tx only 90d group were considered the most relevant . when comparing this group with the control animals , there were no significant differences at 1 hz , but major differences at 5 hz ( p & lt ; 0 . 01 ) and 10 hz ( p & lt ; 0 . 01 ), indicating decreased frequency - dependent depression ( hyper - reflexia ) 90 days after tx . such decreases in frequency - dependent depression were evident to a similar degree after only 30 days following tx ( tx only 30d ) compared to the control group ( p & lt ; 0 . 01 ), suggesting that hyper - reflexia is manifested within 30 days and remains at similar levels in the chronic ( 90 day ) condition . the effects of mbet for 15 days ( tx + ex 15d ), while producing a numerical reduction of 15 % at each frequency , were not statistically significant . however , after 30 days of mbet ( tx + ex 30d ), there were statistically significant increases in frequency - dependent depression ( decreased percent 30 +%) at all frequencies tested , indicating that passive exercise led to a restoration of h - reflex habituation . the effects of longer durations of mbet produced decreases in frequency - dependent depression that were variable at the lower frequencies ( 1 and 5 hz ), but linearly decrementing at 10 hz . for example , percent frequency - dependent depression decreased from 29 % after 30 days ( tx + ex 30d ), to 28 % after 45 days ( tx + ex 45d ), to 22 % after 60 days ( tx + ex 60d ), to 7 % after 90 days ( tx + ex 90d ) of mbet . fig3 is a comparison of a delav of 30 days following tx before testing the h - reflex in untrained rats ( tx only 30d ) compared to those trained for 30 days after tx ( tx + ex 30d ) and to intact animals ( control ). basically , anova shoved a significant difference across groups ( df = 2 , f = 12 . 22 , p & lt ; 0 . 001 ) and frequencies ( df = 3 , f = 50 . 46 , p & lt ; 0 . 001 ), as well as interaction across these two factors ( df = 6 , f = 2 . 24 , p & lt ; 0 . 05 ). tx only induced statistically significant decreases in frequency - dependent depression at 5 hz (** p & lt ; 0 . 01 ) and 10 hz (** p & lt ; 0 . 01 ) compared to control (++). mbet for 30 days induced an increase in h - reflex frequency - dependent depression ( decreased percent ) when comparing the effects of tx (* p & lt ; 0 . 05 ) to tx + ex 30d (+) at both 5 and 10 hz . these results show that the decrement in low frequency - dependent depression ( 1 - 10 hz ) of the h - reflex induced by tx can be alleviated by mbet in a period as short as 30 days . this study shows that passive exercise in spinally transected animals can be used to restore low frequency - dependent depression of the h - reflex . the decrement in hyper - reflexia was evident in a mbet duration - dependent manner in tx rats . the h - reflex is a reliable measure of spinal circuitry that is altered after an sci . this reflex normally undergoes changes with a variety of rhythmic motions such as stepping ( 29 ), walking ( 30 ), running ( 31 ), and pedaling ( 32 ). in animals , the h - reflex has been a valuable tool to measure changes in spinal circuitry in both contusion ( 20 , 33 ) and transection models ( 23 , 24 ). the h - reflex has also been used to assess changes in spinal circuitry in the human , a recent effort concluding that ( 1 ) chronically paralyzed subjects showed suppression of h - reflexes to a lesser extent than able - bodied normals or acutely paralyzed subjects , ( 2 ) those with acute paralysis showed similar h - reflex suppression as those within 40 weeks of their sci , but had decreased frequency - dependent depression after 44 weeks of paralysis ( i . e ., there was marked loss of frequency - dependent depression of the h - reflex in the ‘ chronic ’ condition ), and ( 3 ) changes in muscle fatigue were associated with a decrease in h - reflex suppression over time ( 13 ). the results described herein show that frequency - dependent depression of the h - reflex is decreased in the most ‘ chronic ’ condition tested , 90 days after tx . surprisingly , a similar level of hyper - reflexia was evident even at 30 days ( fig3 ), suggesting that hyper - reflexia assumes a fairly ‘ chronic ’ level within 30 days after tx in the rat . these results are consistent with those of thompson et al . ( 20 ), who reported significantly less frequency - dependent depression of the monosynaptic reflex at 28 and 60 days postinjury in spinal cord contused rats than in normal animals , but this effect was not evident 6 days after injury . such results suggest that the onset of low frequency - dependent depression of the h - reflex takes weeks to develop . further studies are required to determine the time course of the decrement in frequency - dependent depression of the h - reflex in the rat . other forms of exercise in animal models also show promise in the ability to effect changes in lower limb function following sci . for example , adult cats showed recovery of full weight - bearing hindlimb stepping on a treadmill within a few weeks following complete spinal transection if treadmill training was implemented ( 34 , 35 ). completely spinalized cats that were not treadmill - trained were 3 times less likely to step than those that were trained ( 36 ). if spinal cord hemisected rats are immobilized , the expected recovery is delayed and may not be as great ( compared to exercised animals ) once use is restored ( 37 ). we showed that 90 days of mbet minimized loss of muscle mass in transected and exercised , compared to transected only , rats ( 23 , 24 ). additionally , we showed that mbet restored frequency - dependent depression of the h - reflex to normal levels in exercised transected rats ( 24 , 25 ). the h - reflex of nonexercised tx animals failed to show frequency - dependent depression at most stimulation frequencies . the present studies were designed to determine the time course of recovery induced by mbet on low frequency - dependent depression of the h - reflex . mbet for only 15 days , while not inducing a statistically significant effect on h - reflex habituation , did have a numerical effect , increasing frequency - dependent depression ( decreasing percent inhibition ) at 10 hz from 69 to about 42 %. mbet for 30 , 45 , and 60 days induced a similar , significant reduction in percent inhibition from 69 to around 22 - 29 %. not until 90 days of mbet was there a further reduction to around 7 %. these findings suggest that mbet had a fairly rapid ameliorative effect on h - reflex frequency - dependent depression , and could have added effects if continued long term . use of a motorized bicycle exercise trainer to normalize frequency - dependent habituation of the h - reflex in spinal cord injury in an animal model in section 1 of this example , exercise on a motor - driven bicycle was shown to normalize hyperactivity of the h - reflex in plantar muscles of rats after complete spinal cord transection at the t10 level . this study was conducted to test the effect of a similar exercise program in humans with an sci on the habituation of the h reflex to assess hyper - reflexia , a component of spasticity and an indirect assessment of spasticity . the participant was a 24 - year - old male with asia b c7 sci due to a gunshot wound thirteen months prior to the start of this study . his post - injury rehabilitation was complicated by neuropathic pain in the right upper extremity which was mitigated the use of neurontin , and was subsequently discharged to live with his parents . spasticity was a problem for him , which he was managing with routine stretches . he wanted to avoid aggressive management with either oral or intrathecal medication . prior to starting the exercise program , a d - dimer blood test indicated a low risk of a deep vein thrombosis ( dvt ) and a venous doppler ultrasound exam was not required . his risk of osteoporosis was assessed with a standard dexa scan . the participant &# 39 ; s z score at the femoral neck was − 2 . 8 , and risk of fracture was discussed prior to starting the exercise program . the risk and benefits were discussed with him and he signed an informed consent form approved by the irb at uams prior to the start of the study . he was passively exercised 5 days a week for 13 weeks on a motorized bicycle exercise trainer designed for the purpose at the university of arkansas for medical sciences . he performed the exercises from his wheelchair with feet secured to the pedals . the first week was started at a slower speed , approximately 20 cycles per minute ( cpm ), but by the second week he was exercising at 40 cpm for 30 minutes with a 10 minute break and then another 30 minutes . h - reflex habituation was assessed prior to the exercise session with the subject prone with knee slightly flexed and the ankle in 0 degree dorsiflexion . the frequency range tested was 0 . 2 , 1 , 5 , and 10 hz . the stimulating electrode ( all electrodes disposable type , nicloet biomedical , inc .) was positioned over the right tibial nerve at the level of the popliteal fossa , and the recording electrode over the right soleus muscle with the reference electrode over the right achilles tendon . a ground electrode was placed over the right tibial crest . after discarding the first five trials , tent h - reflexes were averaged at each frequency tested . the amplitude of the h - reflex was computed as a percentage of that recorded at low frequency ( 0 . 2 hz ), which was considered to be 100 %. this study was approved by the institutional review board at the university of arkansas for medical sciences . fig4 , panel a shows h - reflex testing on 16 normal controls and 6 sci victims . these two groups were statistically significantly different ( two - way anova , f 1 , 19 = 21 . 2 , p & lt ; 0 . 0001 ) and the nwhan - keuls post - hoc test showed a statistically significant difference in low frequency h - reflex habituation at 5 hz ( p & gt ; 0 . 01 ). recordings from the present subject ( fig4 , panel b ) show the habituation profile for the tested frequencies before mbet ( mbet - 1 pre ) and after 12 weeks of mbet ( mbet - 1 post ). note that this subject &# 39 ; s habituation at both 5 hz and , especially , 10 hz before the mbet treatment was at a higher percentage than average for the other sci patients . fig5 shows the results of h - reflex testing in the present subject throughout the testing period . initially , h - reflex amplitude at 5 hz was 76 % and at 10 hz 65 %, consistent with a hyperactive spastic muscle . after 8 weeks , the amplitude began to decrease and at 12 weeks had decreased to 27 % at 5 hz and 12 % at 10 hz ( comparable to normal controls ). exercise was stopped at 13 weeks , but the subject &# 39 ; s h - reflex was reassessed weekly for four weeks . the amplitude of the h - reflex remained at near normal levels for two weeks after exercise ceasing , but by 17 weeks the h - reflex at 5 hz at increased to 57 % and at 10 hz to 52 %, indicating a rapid return of the subject &# 39 ; s spasticity . a linear component was used to test differences in frequency - dependent h - reflex amplitudes between early and late times in the exercise and testing period . in weeks 1 - 6 the average response was 66 ± 7 % compared to 38 ± 8 % in weeks 10 - 15 . these average values of h - reflex amplitudes are significantly different ( student &# 39 ; s t - test , t = 5 . 70 , p & lt ; 0 . 0009 ). similarly , at 10 hz the average h - reflex amplitude in weeks 1 - 6 was 59 ± 11 % in weeks 10 - 15 ( t = 5 . 21 , p & lt ; 0 . 002 ). these differences indicate the effectiveness of mbet exercise in normalizing the frequency - dependent amplitude of the h - reflex . this subject also reported reduced spastisity at the end of the training regiment , as would be expected due to the normalization of reflexes . modafinil restored frequency dependent depression of the h - reflex in spinally transected rats rats were spinally transected as in section 1 of this example . one group of rats received no exercise after transection . a second group of rats was treated with passive exercise using the motorized bicycle exercise trainer , as in section 1 . passive exercise was begun within 7 days of transection . a third group of transected rats were not exercised but were given 3 mg / kg modafinil per day . a 100 mg modafinil pill was ground by mortar and pestle and mixed with 9 ml of syrup and 1 ml of water . the dose to the animal was 0 . 1 ml p . o . daily ( 3 mg / kg / day ). modafinil was started between 48 hours and 7 days after transection . a fourth group of rats was untransected normal controls . each group contained 9 to 12 rats . at 30 days after transection , the h - reflex of each rat was measured as in section 1 . the h - reflex of the four groups of rats was measured at 30 days after transection at stimulation frequencies from 0 . 2 to 10 hz . the four groups of rats were normal controls ( control ), transected and untreated for 30 days after transection ( tx − 30 ), transected and treated solely with 3 mg / kg / day modafinil p . o . for 30 days ( mod ), and transected and treated solely with passive exercise for 30 days ( mbet 30 ). the results are shown in fig . d 1 . fig6 shows that as you increase the stimulation for 0 . 2 to 10 hz , the h - reflex amplitude in intact rats ( control ) decreases markedly . in rats that were transected and tested 30 days after surgery ( tx − 30 ), the amplitude of the reflex did not habituate — that is , there was hyper - reflexia . in rats that were transected and mbet begun within 7 days after surgery and exercised for 30 days ( mbet 30 ), the reflex amplitude decreased as in intact animals , indicating that passive exercise led to a normalization of the hyper - reflexia . in animals that were transected and not exercised but given daily doses of modafinil beginning 7 days after surgery ( mod ), the reflexes were normalized , indicating that modafinil also alleviated hyper - reflexia . hyper - reflexia is a component of spasticity , so that normalization of hyper - reflexia indicates a reduction or elimination of spasticity . these studies demonstrated that oral administration of modafinil , which is used to promote waking in narcoleptic subjects , was effective in reducing hyper - reflexia after spinal transection a novel molecular mechanism for hyper - reflexia . we reasoned that a delay of weeks to months in the onset of hyper - reflexia and spasticity indicated that a mechanism involving protein expression may be at play . if changes in presynaptic inhibition were really responsible for hyper - reflexia and spasticity , their onset would be immediate after injury , not delayed . we also reasoned that , following a spinal cord lesion , the tissue below the level of the lesion would regress to a neonatal state . a major change in development is the change in electrical coupling between motoneurons . in the developing rat spinal cord , motoneurons are electrically coupled until about 7 days of life , but coupling disappears by 14 days of life ( 52 ). electrical coupling in neurons depends on gap junctions , specifically , the gap junction protein connexin 36 ( cx36 ) ( 53 ). we used rt - pcr to detect changes in cx36 expression after tx . we sampled spinal cord tissue below the level of the transection after testing the h - reflex . fig7 shows that cx36 expression decreased dramatically by 30 days after tx ( tx30d ) compared to intact rats ( normal ). in rats that had 30 days of mbet after transection ( tx + mbet30d ), cx36 expression increased significantly above the 30 day tx only rats . this suggests that cx36 expression a ) decreased after tx , and b ) increased towards the levels in intact animals after tx + mbet . these results suggest that , following tx , motoneurons may become electrically uncoupled due to decreases in gap junctions after injury . mbet , perhaps by inducing alternation in both limbs simultaneously , thereby mimicking bipedal locomotion , may lead to recoupling of motoneurons leading to coordinated activation , rather than independent activation , of motor units , which is facilitated by an increase in gap junctions . these results suggest that changes in cx36 levels in spinal cord tissue , in response to tx , could play a role in the development of hyper - reflexia and spasticity . since we suspected that gap junctions may be involved in the manifestation of hyper - reflexia , we reasoned that an agent that would increase electrical coupling in neurons might be useful for the treatment of hyper - reflexia , the main component of spasticity . recent unpublished evidence from rodolfo llinas at new york university suggests that modafinil increases electrical coupling in cortical cells , inferior olivary cells and thalamic reticular cells ( personal communication ). we reasoned that modafinil could increase electrical coupling in spinal cord neurons , and so began the study described above using modafinil as a potential treatment for hyper - reflexia . in summary , the previously unknown mechanism of action of modafinil may be to increase electrical coupling in brain cells involved in waking , making it effective for the treatment of narcolepsy . such a mechanism has never before been proposed but will be published soon by the llinas laboratory . since we discovered that hyper - 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