Treating neuropathic pain in spinal cord injured individuals

Abstract

<DP=1>²Methods of treating neuropathic pain in spinal cord injured individuals by ²administering ²levetiracetam or brivaracetam are described, including the treatment of ²neuropathic pain ²perceived by an individual to be below the level of spinal cord injury, ²including wherein the pain ²is generated by spinal cord tissue caudal to the level of the spinal cord ²injury.²

Description

<DP=1>²WO 2018 / 071893 PCT / US2017 / 056745²TREATING NEUROPATHIC PAIN IN SPINAL CORD INJURED INDIVIDUALS²PRIORITY²

[0001] The patent application claims priority to U.S. Provisional Patent ²Application No. ²62 / 408,490 filed on October 14, 2016.²FIELD²

[0002] Disclosed herein are methods of treating neuropathic pain in spinal ²cord injured²individuals by administering levetiracetam or brivaracetam.²BACKGROUND²

[0003] Spinal cord injury (SCI) frequently results in severe or disabling ²pain. Individuals with a ²SCI suffer anywhere from between 20 and 77% incidence of some level of severe ²or disabling ²chronic pain. Davis et al., Clin Orthop 112:76-80 (1975); Richards et al., ²Pain 8:355-366 (1980); ²Siddall et al., Spinal Cord 39:63-73 (2001); Stormer et al., Spinal Cord 446-²455 (1997); Woolsey, ²J Am Paraplegia 9:39-41 (1986). Patients suffering from at least some level of ²severe or disabling ²chronic pain show reduced rehabilitation potential and tend to have a ²significant overall reduction ²in quality of life.²

[0004] Central pain has proven notoriously difficult to treat, often proving ²recalcitrant to modern ²medical and surgical pain treatment procedures. Surgical treatment of specific ²dorsal root entry ²zone(s) (DREZ(s)) of the spinal cord are of particular interest. Surgical ²treatment of a central pain ²generating DREZ at the level of an injury is believed to disrupt the neural, ²i.e., electrical, ²communication and / or generation of aberrant pain signals that result from the ²injury. Initially, ²empirical techniques have been used to target DREZ sites for surgical ²treatment, resulting in ²modest outcomes for the patient, i.e., DREZ sites at the site of injury ²targeted for treatment. ²Friedman et al., J Neurosurg 65:465-469 (1986); lshijima et al., Appl ²Neurophysiol 51:2-5, ²175-187 (1988); Rath et al., Acta Neurochir, 138:4, 364-369 (1996); Rath et ²al., Sterotact Funct ²Neurosurg 68:1-4, Pt 1, 161-167 (1997). One of the more relevant patient ²studies using this ²empirical technique suggests that approximately 50% of patients so treated ²achieve good relief ²from SCI associated pain. Friedman et al., J Neurosurg. 65:465-469 (1986). In ²that series, at-level ²pain, i.e., pain at the immediate vicinity of the injury, responded best (74% ²"good results") and ²below-level pain, i.e., pain below the level of injury, responded poorly (20% ²"good results").²1²Date Recue / Date Received 2022-04-14²<DP=2>²CA 03040482 2019-04-12²WO 2018 / 071893 PCT / US2017 / 056745²

[0005] Anti-epileptic drugs are often suggested for treating neuropathic pain. ²Two of these ²commercially approved compounds, levetiracetam and brivaracetam, are 2-oxo-²pyrrolidin-1-y1 ²bytanamide derivatives that act by binding to synaptic vesicle glyooprotein 2A ²(SV2A). However, ²this class of compounds has been disparaged as a method of treating pain in ²SCI individuals. ²Finnerup et al., Scandinavian Journal of Pain 1, S1 (2009) S3-S11, described ²levetiracetam as ²ineffective in treating pain resulting from spinal cord injury. According to ²Finnerup et al., ²"levetiracetam does not relieve neuropathic pain or spasm severity following ²spinal cord injury." ²Finnerup et al. further pointed out that the lack of efficacy on the ²postmastectomy syndrome ²suggests a lack of interference with mechanisms underlying peripheral ²postsurgical neuropathic ²pain as well. Finnerup et al. suggested that it is possible that the ²pharmacological action of ²levetiracetam is not involved in neuropathic pain mechanisms and that ²interference with the SV2A ²in levetiracetam doses used in humans will have no impact on neuropathic pain.²

[0006] Levetiracetam has been shown to have no analgesic or other benefit in ²patients with ²neuropathic pain following SCI. This suggests that the same would be true for ²any SV2A-affecting ²compound, including brivaracetam. (Finnerup et al.).²SUMMARY²

[0007] Contrary to the general understanding in the field, the disclosure ²shows that there is a ²strong correlation between SV2A and SCI neuropathic pain.²

[0008] In one aspect, the disclosure is directed to methods of treating ²neuropathic pain in a spinal ²cord injured individual in need thereof. A 2-oxo-pyrrolidin-1-ylbytanamide ²derivative is ²administered to the Individual.²

[0009] In some aspects, the 2-oxo-pyrrolidin-1-ylbytanamide derivative is ²levetiracetam ²((S)-2-(2-oxopyrrolidin-1-0butanamide). In other variations, 2-oxo-pyrrolidin-²l-ylbytanamide ²derivative is brivaracetam (2-(2-oxo-4-propylpyrrolidin-1-Abutanamide).²

[0010] In some aspects, the neuropathic pain is perceived by the patient to be ²below the level of ²the spinal cord injury. In some aspects, the neuropathic pain is perceived by ²the patient to be ²below the neurological level of the spinal cord injury. In some variations, ²the pain is generated by ²spinal cord tissue caudal to the level of the spinal cord injury. In some ²variations, thepain is ²generated by spinal cord tissue cephalad to the level of the spinal cord ²injury.²

[0011] The neuropathic pain drug can be administered below the level of the ²spinal cord injury. ²Further, the 2-oxo-pyrrolidin-1-ylbytanamide derivative can be administered ²intrathecally, below ²the level of spinal cord injury.²2²<DP=3>²CA 03040482 2019-04-1.2²WO 2018 / 071893 PCT / US2017 / 056745²BRIEF DESCRIPTION OF THE DRAWINGS²

[0012] The following drawings are made to illustrate aspects of the present ²disclosure, and are ²not intended to be limiting:²

[0013] Figure 1 depicts a box plot representation of SV2A, according an ²illustrative embodiment; ²

[0014] Figure 2 depicts a preoperative sensory chart and regions of perceived ²below-level pain, ²according to an illustrative embodiment;²

[0015] Figure 3 depicts normal DREZ recording unfiltered, filtered data ²(spindles) and FFT ²labeled data, according to an illustrative embodiment;²

[0016] Figure 4 depicts electrically hyperactive DREZ recording showing an ²increase in ²peak-to-peak voltage of original recording, increase in spindles, and skewing ²of high electrical ²activity across frequency spectrum compared to Figure 3, according to an ²illustrative ²embodiment;²

[0017] Figure 5 depicts a post-microcoagulation DREZ recording of a patient in ²which the ²frequency plot is skewed toward lower frequencies and absence of spindles ²compared to Figure ²4, according an illustrative embodiment;²

[0018] Figure 6 depicts 12 weighted MRI showing regions of spinal cord ²transection and regions ²of DREZ microcoagulation of a patient, according an illustrative embodiment;²

[0019] Figure 7 depicts postoperative regions of pain relief in patients, ²according to an illustrative ²embodiment;²

[0020] Figure 8 depicts a cross section represents the L1 spinal cord at or ²immediately cephalad ²to the level of SCI, of a patient with both at-level inguinal region and below-²level foot pain and ²DREZ hyperactivity solely at L1 (cross-hatch), according to an illustrative ²embodiment.²

[0021] Figure 9 depicts normal electrical activity of the DREZ of 110 in ²regions below the cervical ²level of SCI in a complete quadriplegic patient with leg pain, according an ²illustrative embodiment; ²

[0022] Figure 10 depicts electrical hyperactivity in the DREZ of 111 below the ²cervical level of ²complete spinal cord injury, according an illustrative embodiment;²DETAILED DESCRIPTION²

[0023] This disclosure relates to using levetiracetam or brivaracetam to treat ²neuropathic pain in ²spinal cord injured individuals. Pain-producing tissues correlating with ²sympathetically mediated ²central neuropathic pain in SCI patients were acquired. Comparative analysis ²of molecular ²markers between electrically hyperactive pain-producing tissue and ²electrically normal non-pain²3²<DP=4>²CA 03040482 2019-04-12²WO 2018 / 071893 PCT / US2017 / 056745²producing tissue showed an upregulation of SV2A in the pain-producing tissue ²implicated in ²sympathetically mediated central pain. Contrary to the prevailing view, the ²SV2A is implicated in ²spinal cord injury neuropathic pain. As such, drugs such as levetiracetam or ²brivaracetam that act ²on SV2A can be used to treat SCI pain, including sympathetically medicated ²central pain ²perceived below the neurological level the SCI. Pain includes neuropathic, SCI ²pain, and central ²pains referring to their central nervous system origin.²

[0024] Methods of treating (i.e. alleviating, reducing, diminishing or ²otherwise attenuating) ²neuropathic pain in a patient in need thereof by administering 2-oxo-²pyrrolidin-1-y1 bytanamide ²derivatives such as levetiracetam or brivaracetam are described herein.²

[0025] Relieving SCI neuropathic pain has become an extremely difficult ²problem with either ²pharmacological or surgical treatment. It has generally been believed that ²generators of SCI pain ²must come solely from spinal cord regions above (cephalad) the level of SCI or ²from the brain ²itself. Extreme surgical measures such as complete spinal cord transection ²have been performed ²to eliminate all possible influence of the spinal cord below the level of ²injury in these severe and ²pharmacologically refractory pains.²

[0026] It has now been discovered that spinal cord below (caudal) the level of ²injury can, in fact, ²generate below-level SCI neuropathic pain and be its sole source. In ²particular, eradicating spinal ²cord regions of hyperactivity below the level of injury within the dorsal grey ²matter (Rexed layers ²1-3) can result in complete pain relief, even if the spinal cord had been ²completely transected. ²

[0027] Spinal cord pain can be mediated through the sympathetic nervous ²system. ²Specifically,hyperactive electrical neuronal signals originating in spinal ²cord below the level of ²injury can be routed around the injury site by way of the sympathetic chain to ²reach brain pain ²centers. The pain is be perceived as below the neurological level of spinal ²cord injury, where it ²should not be perceived. For example, in a complete spinal cord injury at the ²neurological level of ²110, a patient should not feel lower (caudal) pain from the umbilicus. ²However, the patient can ²nevertheless perceive pain in these regions.. The pain can be generated by ²tissues located ²below (caudal to) the level of spinal cord injury or above (cephalad to) the ²level of spinal cord ²injury. Such sympathetically mediated spinal cord injury pain is perceived by ²the patient to be ²below the neurological level of spinal cord injury (referred to herein as ²"below-level pain"). ²

[0028] Contrary to expectations in the art, SV2A has been discovered to ²correlate with ²below-level pain. Specifically, SV2A is markedly upregulated in electrically ²hyperactive spinal ²cord tissue caudal to the level of injury and / or cephalad to the level of ²injury, and eradication of ²this hyperactive tissue can result in complete pain relief, even if the spinal ²cord had been ²completely transected prior. As such, SV2A, with its increase in expression ²after spinal cord²4²<DP=5>²CA 03040482 2019-04-12²WO 2018 / 071893 PCT / US2017 / 056745²injury, has been identified as a generator of neuropathic pain perceived below ²the neurological ²level of spinal cord injury (sympathetically mediated pain).²

[0029] Contrary to conventional wisdom, levetiracetam and brivaracetam can ²relieve below-level ²neuropathic pain (sympathetically mediated pain) with oral, SC, IV, ²sublingual, intra, or intrathecal ²administration. Drugs associated with below level pain, including ²levetiracetam or brivaracetam, ²can be used to treat pain, including pain perceived as below the neurological ²level of spinal cord ²injury.²Definitions²

[0030] "Pharmaceutically acceptable vehicle" refers to a pharmaceutically ²acceptable diluent, a ²pharmaceutically acceptable adjuvant, a pharmaceutically acceptable excipient, ²a ²pharmaceutically acceptable carrier, or a combination of any of the foregoing ²with which a ²compound provided by the disclosure may be administered to a patient and which ²does not ²destroy the pharmacological activity thereof and which is non-toxic when ²administered in doses ²sufficient to provide a therapeutically effective amount of the compound.²

[0031] "Pharmaceutical composition" refers to levetiracetam or brivaracetam ²and at least one ²pharmaceutically acceptable vehicle with which levetiracetam or brivaracetam ²is administered to ²a patient.²

[0032] "Therapeutically effective amount" refers to the amount of a compound ²that, when ²administered to a subject for treating a disease, or at least one of the ²clinical symptoms of a ²disease, is sufficient to affect such treatment of the disease or symptom ²thereof. The ²"therapeutically effective amount" may vary depending, for example, on the ²compound, the ²disease and / or symptoms of the disease, severity of the disease and / or ²symptoms of the disease ²or disorder, the age, weight, and / or health of the patient to be treated, as ²well as the judgment of ²the prescribing physician. An appropriate amount in any given instance may be ²ascertained by ²those skilled in the art or capable of determination by routine ²experimentation.²

[0033] "Therapeutically effective dose" refers to a dose that provides ²effective treatment of a ²disease or disorder in a patient. A therapeutically effective dose may vary ²from compound to ²compound, and from patient to patient, and may depend upon factors such as the ²condition of the ²patient and the route of delivery. A therapeutically effective dose may be ²determined in ²accordance with routine pharmacological procedures known to those skilled in ²the art.²Compounds²

[0034] Levetiracetann and brivaracetam are antiepileptic medications. Both ²levetiracetam and ²brivaracetam are 2-oxo-pyrrolidin-1-ylbytanannide derivatives. Levetiracetam, ²or²<DP=6>²CA 03040482 2019-04-12²WO 2018 / 071893 PCT / US2017 / 056745²(S)-2-(2-oxopyrrolidin-1-yl)butanamide, is a single enantiomer prescribed as a ²treatment for ²certain types of seizures. Brivaracetam, or 2-(2-oxo-4-propylpyrrolidin-1-²yl)butanamide, is an ²analog of levetiracetam.²

[0035] Both levetiracetam and brivaracetam bind to SV2A, a known target of ²anti-epileptic drugs. ²Though suggested as drugs that can be used to treat neuropathic pain, ²levetiracetam has been ²believed not to have an effect on pain in SCI patients, including pain below ²the level of the site of ²injury.²

[0036] The disclosure is directed to treating pain in spinal cord injured ²individuals. In some ²variations, levetiracetam or brivaracetam is administered to an individual to ²treat neuropathic ²pain. In additional variations, levetiracetam or brivaracetam is administered ²to an individual to ²treat neuropathic pain in a spinal cord injured individual. In further ²variations, levetiracetam or ²brivaracetam is administered to treat sympathetically mediated central nervous ²system pain in a ²spinal cord injured individual. In still further, the neuropathic pain can be ²sympathetically ²mediated. In additional variations, the neuropathic pain is perceived below ²the neurological level ²of injury. In additional variations, pain generating spinal cord tissue ²originates below the level of ²the location of spinal cord injury.²Pharmaceutical Compositions²

[0037] Pharmaceutical compositions provided by the disclosure may comprise a ²therapeutically ²effective amount of levetiracetam or brivaracetam together with a suitable ²amount of one or more ²pharmaceutically acceptable vehicles so as to provide a composition for proper ²administration to ²an individual. Suitable pharmaceutical vehicles are described in the art.²

[0038] In certain embodiments, levetiracetam or brivaracetam may be ²incorporated into ²pharmaceutical compositions to be administered intrathecally. Oral ²compositions may be ²prepared in a manner known in the pharmaceutical art and comprise ²levetiracetam or ²brivaracetam and at least one pharmaceutically acceptable vehicle. Oral ²pharmaceutical ²compositions may include a therapeutically effective amount of levetiracetam ²or brivaracetam and ²a suitable amount of a pharmaceutically acceptable vehicle, so as to provide ²an appropriate form ²for administration to an individual.²

[0039] Pharmaceutical compositions comprising levetiracetam or brivaracetam ²may be ²manufactured by conventional mixing, dissolving, granulating, draoee-making, ²levigating, ²emulsifying, encapsulating, entrapping, or lyophilizing processes. ²Pharmaceutical compositions ²may be formulated in a conventional manner using one or more physiologically ²acceptable ²carriers, diluents, excipients, or auxiliaries, which facilitate processing of ²the compounds and one²6²<DP=7>²CA 03040482 2019-04-12²WO 2018 / 071893 PCT / US2017 / 056745²or more pharmaceutically acceptable vehicles into formulations that can be ²used ²pharmaceutically. Proper formulation is dependent upon the route of ²administration chosen. ²Pharmaceutical compositions provided by the disclosure may take the form of ²solutions, ²suspensions, emulsion, tablets, pills, pellets, capsules, capsules containing ²liquids, powders, ²suppositories, emulsions, aerosols, sprays, suspensions, or any other form ²suitable for ²administration to an individual.²

[0040] Pharmaceutical compositions are suitable for parenteral administration, ²including ²intrathecal injection. The pharmaceutical can be a sterile injectable ²preparation of levetiracetam ²or brivaracetam in, for example, a solution which is isotonic with the blood ²or cerebrospinal fluid of ²the recipient. Useful formulations also comprise concentrated solutions or ²solids containing the ²active ingredient which upon dilution with an appropriate solvent give a ²solution suitable for ²parenteral (including intrathecal) administration. The parenteral compositions ²include aqueous ²and non-aqueous formulations which may contain conventional adjuvants such as ²buffers, ²bacteriostats, sugars, thickening agents and the like. The compositions may be ²presented in unit ²dose or multi-dose containers, for example, sealed ampules and vials.²

[0041] Levetiracetam or brivaracetam may be incorporated into pharmaceutical ²compositions for ²administration by any other appropriate route of administration including ²p.o.(by mouth), IV ²(intravenously), SC (subcutaneously), IC (intrathecally - spinal fluid), ²intranasally, and ²sublingually.²

[0042] Pharmaceutical compositions provided by the disclosure may be ²formulated in a unit ²dosage form. A unit dosage form refers to a physically discrete unit suitable ²as a unitary dose for ²individuals undergoing treatment, with each unit containing a predetermined ²quantity of ²levetiracetam or brivaracetam calculated to produce an intended therapeutic ²effect. A unit dosage ²form may be for a single daily dose, for administration 2 times per day, or ²one of multiple daily ²doses, e.g., 3 or more times per day. When multiple daily doses are used, a ²unit dosage form may ²be the same or different for each dose. One or more dosage forms may comprise ²a dose, which ²may be administered to an individual at a single point in time or during a ²time interval.²

[0043] The amount of a levetiracetam dose can be chosen to any quantity ²appropriate for a ²specific treatment. In some variations, the levetiracetam dose can be at least ²20 mg, 40 mg, 60 ²mg, 80 mg, 100 mg, 120 mg, 140 mg, 160 mg, 180 mg, and 200 mg. In some ²variations, the dose ²can be less than or equal to 300 mg, 280 mg, 260 mg, 240 mg, 220 mg, 200 mg, ²180 mg, 160 mg, ²140 mg, 120 mg, and 100 mg.²

[0044] In some variations, levetiracetam can be administered by intravenous ²infusion at a daily ²dose of 1000 mg. The daily dose can be administered as a twice daily 500 mg ²dose. In some²7²<DP=8>²CA 03040482 2019-04-12²WO 2018 / 071893 PCT / US2017 / 056745²variations, the dose can increase in increments of 1000 mg / day every two weeks ²to a maximum ²daily dose of 3000 mg. In some variations, levetiracetam can be administered ²in 1000 mg once ²daily doses. In some variations, levetiracetam can be administered in ²increments of 1000 mg ²every two weeks to the maximum daily dose of 3000 mg. In some variations, the ²daily dose of ²levetiracetam can be administered as an oral solution of 100 mg / ml. In some ²variations, ²levetiracetam can be administered as a 250 mg, 500 mg, 750 mg, or 1 gram ²tablet.²

[0045] In some variations, levetiracetam can be administered intrathecally, ²such as with an ²infusion pump (e.g., the Medtronic Synchromed II Programmable Infusion Pump). ²Intrathecal ²administration can has lower dosing than other dosing methods, and can be ²delivered ²continuously, as a bolus, or with varying concentration over a period of time. ²In some variations, ²the levetiracetam intrathecal dose can be at least 20 micrograms, 40 ²micrograms, 60 micrograms, ²80 micrograms, 100 micrograms, 120 micrograms, 140 micrograms, 160 micrograms, ²180 ²micrograms, and 200 micrograms. In some variations, the dose can be less than ²or equal to 300 ²micrograms, 280 micrograms, 260 micrograms, 240 micrograms, 220 micrograms, ²200 ²micrograms, 180 micrograms, 160 micrograms, 140 micrograms, 120 micrograms, ²and 100 ²micrograms.²

[0046] In some variations, the brivaracetam dose can be at least 10 mg, 25 mg, ²50 mg, 75 mg, ²100 mg, 125 mg, 150 mg, 175 mg, or 200 mg. In some variations, the dose can be ²less than or ²equal to 300 mg, 275 mg, 250 mg, 225 mg, 200 mg, 175 mg, 150 mg, 125 mg, or ²100 mg. In some ²variations, brivaracetam can be administered as an oral solution of 100 mg / mL. ²Such variations ²can be by any mode of administration, including intravenous and oral ²administration. ²Brivaracetam can be administered as 50 mg PO BID initially. Based on ²individual tolerability and ²therapeutic responses, the dose can be adjusted down to 25 mg BID (50 mg / day) ²or up to 100 mg ²BID (200 mg / day). Alternatively, brivaracetam can be administered by IV ²injection. Injection can ²be at the same dosage and same frequency as tablets or oral solution. In some ²variations, ²brivaracetam can be administered intravenously at 50 mg / 5mL. In some ²variations, brivaracetam ²can be administered by oral tablets at 10 mg, 25 mg, 50 mg, 75 mg, or 100 mg.²

[0047] In some variations, brivaracetam can be administered intrathecally. As ²with intrathecal ²levetiracetam administration, administration can has lower dosing than other ²dosing methods, ²and can be delivered continuously, as a bolus, or with varying concentration ²over a period of time. ²In some variations, the brivaracetam dose can be at least 10 micrograms, 25 ²micrograms, 50 ²micrograms, 75 micrograms, 100 micrograms, 125 micrograms, 150 micrograms, 175 ²²micrograms, or 200 micrograms. In some variations, the dose can be less than ²or equal to 300²8²<DP=9>²WO 2018 / 071893 PCT / US2017 / 056745²micrograms, 275 micrograms, 250 micrograms, 225 micrograms, 200 micrograms, ²175 ²micrograms, 150 micrograms, 125 micrograms, or 100 micrograms.²

[0048] An appropriate dose of levetiracetam or brivaracetam or pharmaceutical ²composition ²comprising levetiracetam or brivaracetam may be determined according to any ²one of several ²well-established protocols. For example, animal studies such as studies using ²mice, rats, dogs, ²and / or monkeys may be used to determine an appropriate dose of a ²pharmaceutical compound. ²Results from animal studies may be extrapolated to determine doses for use in ²other species, ²such as, for example, humans²

[0049] In some variations, brivaracetam or levteracetam can be administered as ²recommended ²by FDA guidance.²

[0050] Brivaracetam can be administered as an intravenous solution at 10 ²mg / mL, a 10 mg / mL ²oral solution, or as tablets. For tablet or oral solution administration, the ²recommended starting ²dosage is 50 mg twice daily. Based on individual patient tolerability and ²therapeutic response, the ²dosage may be adjusted down to 25 mg twice daily (50 mg per day) or up to 100 ²mg twice daily ²(200 mg per day). When oral administration is not feasible, the same dosage ²can be administered ²by intravenous injection. The dosage of brivaracetam can be altered from the ²approved dosage. ²

[0051] Levteracetam can be administered orally at 1000 mg daily as an oral ²immediate release or ²oral extended release. Levteracetam can be administered by intravenous ²injection when oral ²administration is not feasible. The dosage of levteracetam can be altered from ²the approved ²dosage.²

[0052] Either brivaracetam or levteracetam can be administered in varying ²amounts lower or ²higher than described herein without departing from the scope of the ²disclosure.²EXAMPLES²

[0053] The following examples provide support for aspects of the disclosure. ²They are not ²intended to be limiting. To the contrary, the examples can be varied in ²keeping with the spirit of the ²disclosure.²Example 1: Tissues from Spinal Cord Injured Individuals²

[0054] Tissues implicated in spinal cord pain were acquired and fast frozen. ²Methods of acquiring ²tissues that that exhibit spinal cord pain are have been described, for ²example, in U.S. Patent No. ²8,694,107.²

[0055] Spinal cord tissues producing central pain were acquired surgically. ²First, the spinal cord ²tissue implicated in perceived pain was determined by comparing the anatomical ²location of ²perceived pain to a somatotopic map of SCI neuropathic pain generating spinal ²cord tissue.²9²Date Recue / Date Received 2022-04-14²<DP=10>²WO 2018 / 071893 PCT / US2017 / 056745²Spinal cord tissue displaying hyperactive electrical signals were then ²identified in these spinal ²cord regions by measuring electrical hyperactivity of tissues. Non-²electrically hyperactive, ²non-pain generating DREZ sites, were identified in the same individual. ²Samples of both the ²hyperactive, pain generating DREZ SPINAL CORD TISSUE and non-hyperactive, non-²pain ²generating DREZ spinal cord tissue was taken from the individual.²

[0056] Tissues were obtained surgically exposing the DREZ tissue inclusive of ²the substantia ²gelatinosa tissue above (cephalad), below (caudal), and at the level of ²injury. A recording ²electrode was inserted approximately 2 mm deep into the dorsal grey matter of ²the spinal cord, ²entering at the dorsal root entry zone (DREZ). Recordings of spontaneous ²electrical activity were ²recorded for one second.²

[0057] Recordings were performed bilaterally along the DREZs cephalad and ²caudal to the level ²of the injury approximately 1 mm apart The recordings were analyzed using fast ²Fourier ²transform (FFT) root Mean Square analysis and "spindle" analysis (as disclosed ²in U.S. Patent ²Publication Nos. 2010 / 0203022 and 2007 / 0016264) to identify regions of ²neuroelectrical ²hyperactivity. The regions recorded correspond to Rexed layers 1, 2, and 3 ²within the dorsal ²grey matter.²

[0058] The recordings were guided by a somatotopic map, and made both cephalad ²and caudal ²to the level of injury as well as at the level of the injury until no ²additional electrical hyperactivity ²was detected. A small cut in the pia was made and a micro-pituitary rongeur ²was inserted to ²remove an approximate lx1x2 mm piece of dorsal grey matter. The tissue was ²identified as ²pain-producing "hyperactive" or non-pain producing "normoelectric" based on ²the electrical ²recordings. The tissue was snap-frozen within 10 minutes of excision and ²stored at - 81 C. ²

[0059] Tissue is acquired both from hyperactive (i.e., pain generating) and ²non-hyperactive (i.e., ²non-pain generating) DREZ sites to determine the difference in protein ²expression. Expressed ²proteins are markers of spinal cord injury pain.²

[0060] Tissue from hyperactive and non-hyperactive spinal cord was then tested ²against a 4,000 ²marker panel, including for SV2A.²Example 2: SV2A Expression²

[0061] The tissue was extracted using T- - Per tissue protein extraction agent ²(Thermo Scientific) ²per the manufacturer's recommendation. 200 pL of buffer plus Halt protease ²inhibitor cocktail ²(Pierce Part# 78430) was added. The tissue was homogenized in a tube on ice ²with a rotary ²pestle for 30 seconds until no tissue fragments were visible. The sample was ²centrifuged at ²>14,000x g for 10 minutes while at 4 C. The supernatant was filtered through a ²0.2 micron filter² Date Recue / Date Received 2022-04-14²<DP=11>²WO 2018 / 071893 PCT / US2017 / 056745²into a sterile tube or plate while at 0 C Millipore Multiscreen GV filter ²plate, 0.22 pm, sterile, Part # ²MSGV2210 or similar). The amount of total protein was determined using Micro ²BCA Protein ²Assay Kit (Thermo Scientific). Aliquots were stored at - 81 C.²

[0062] The tissues were tested by SOMAscare analysis. SOMAscan tests tissue ²against a 4,000 ²protein panel (including SV2A), and a range of concentrations. SOMAscae and ²related methods ²and reagents are described, for example, at U.S. Patent Nos, 5,843,653; ²5,853,984; 5,989,823; ²6,261,783; 6,329,145; 6,531,286; 6,670,132; 6,673,553; 6,706,482; 7,709,192; ²7,855,054; ²7,964,356; 8,975,026 and 8,945,830.²

[0063] Hyperactive pain producing tissue was compared to non-pain producing ²tissue. Targets ²that were upregulated in hyperactive (pain producing) tissue as compared to ²non-pain producing ²tissue were identified as pain targets. These pain targets were analyzed for ²drugs effective ²against the target.²

[0064] A box plot representation of SV2A is depicted in Figure 1. The left box ²shows the RFU ²values of SV2A protein expression in the control group of electrically normal ²non-pain producing ²spinal cord tissue from a patient with neuropathic pain perceived below the ²neurological level of ²SCI. The horizontal line in the control group is the median RFU value of SV2A ²protein expression ²of electrically normal non-pain producing spinal cord tissue in the control ²group. The right box ²represents the measured RFU values of SV2A protein expression of electrically ²hyperactive ²pain-producing spinal cord tissue acquired from the same patient with below-²level spinal cord ²injury neuropathic pain. The horizontal black line in the pain-producing group ²depicts the median ²RFU value of SV2A protein expression of electrically hyperactive pain-²producing spinal cord ²tissue acquired from the same patient with below-level spinal cord injury ²neuropathic pain. ²

[0065] Based on the compared SV2A expression, SV2A shows a 4.6 fold increase ²in expression ²in the pain-producing tissue. The ratio of SV2A in hyperactive substantia ²gelatinosa tissue ²compared to normo-active substantia gelatinosa tissue was 4.6. The q-value of ²the RFU analysis ²was 0.0064.²

[0066] The hyperactive spinal tissue showed an increase in SV2A concentration ²over ²non-hyperactive spinal tissue. Surgical destruction of this hyperactive tissue ²resulted in complete ²relief of below-level neuropathic pain. SV2A is therefore a target for ²treating neuropathic pain in ²spinal cord injured individuals, particularly those with sympathetically ²mediated central pain or ²pain below the level of spinal cord injury.²

[0067] SV2A expression in pain-producing cord therefore correlates with ²neuropathic pain. ²Synaptic vesicle giycoprotein 2A is a ubiquitous synaptic vesicle protein that ²in humans is ²encoded by the S1 / 2,4 gene.²11²Date Recue / Date Received 2022-04-14²<DP=12>²CA 03040482 2019-04-12²WO 2018 / 071893 PCT / US2017 / 056745²Example 3²

[0068] Three patients were aged 49, 54, and 45 years. All were male. All ²sustained thoracic ²spinal cord injuries. All experienced severe below-level neuropathic pain ²(Figure 2). Below-level ²pains were first experienced at 19 years, 2 months, and 2 months post injury, ²respectively. Time ²to surgery from onset of significant below-level pain was 2 years, 8 years, ²and 13 years, ²respectively. Descriptors characterizing the pains were "sharp", "burning", ²and "electrical" (Table ²1)²Table 1²Patient Age ASIA Years with Pain²Classification Severe Pain Descriptors²1 49 T5 A 2 burning²2 54 T4 A 6 Burning²3 45 T3 A 13 Burning,²stabbing, ²electrical, ²pins-needles²

[0069] All three patients had undergone previous surgery to the spinal cord. ²One of the three ²patients had undergone previous spinal cord untethering surgery with expansion ²duraplasty and ²cyst shunting, in a failed attempt to alleviate his below-level pain. The ²other two patients had ²previously undergone DREZ microcoagulation at and cephalad to the level of ²injury in an attempt ²to alleviate SCI at-level and below- level neuropathic pain, achieving ²complete relief only of ²at-level pain. No long term below-level pain relief was achieved in any of the ²three. AD three ²patients were noted to have essentially complete traumatic spinal cord ²transections at the injury ²site, visually confirmed during the previous surgeries to the spinal cords ²described above. During ²these surgeries, a thin band of avascular scar found at the injury site ²replacing normal cord tissue, ²was transected, resulting in visually clear and separate ascending and ²descending portions of the ²spinal cord (Figure 6). Four or more months subsequent to these prior ²surgeries, all three patients ²undeiwent DREZ microcoagulation to the spinal cord, caudal to the region of ²spinal cord ²transection at the injury site, to treat below-level pain. DREZ ²microcoagulation was guided by ²operative electrophysiological monitoring of the DREZ for electrical ²hyperactivity.²

[0070] Pain was experienced continuously in all three patients with bursts in ²intensity occurring ²multiple times daily. Pain was exacerbated by any noxious stimulus to the body ²such as a skin ²sore, or urinary tract infection. Pain occurred in regions of the body in ²which sensation was absent²12²<DP=13>²WO 2018 / 071893 PCT / US2017 / 056745²to testing. All three patients rated their pain as a 10 on a scale of Ito 10, ²10 considered near ²suicidal- level pain.²

[0071] All patients underwent preoperative plain radiography, CT scanning, and ²MR imaging to ²evaluate the spine and spinal cord. Any patient with suicidal ideation ²underwent preoperative ²psychological evaluation and received clearance. Preoperatively all patients ²had undergone ²extensive pharmacological treatment including administration of oral ²antidepressant, anti-seizure, ²and narcotic medications. In general, these medications were considered ²ineffective, although ²"took the edge off' in one patient.²

[0072] Methods of DREZ recording were described in a previous publication and ²were used in ²this study(Falci S, Best L, Bayles R, Lammertse D, Starnes C. J Neurosurg ²Spine 97:193-2000, ²2002) Multilevel laminectomies were performed to expose the spinal cord at the ²injury site and ²level of spinal cord transection, as well as all spinal cord regions more ²caudal, to the level of the ²conus medul lads, Seven to eight laminectomies were performed in each of the ²three patients. ²Spinal levels were determined by intraoperative radiology evaluation or ²knowledge of levels of ²existing spinal instrumentation. lntraoperative ultrasonography was used to ²identify the level of ²spinal cord injury and region of spinal cord transection. The dura mater was ²opened and the ²DREZs identified using microscopic technique, Electrophysiological analyses of ²the DR EZs were ²then performed from the injury site to the tip of the conus medullaris. To ²conduct such analyses, ²an active electrode was inserted into the specific DREZ. The active electrode ²was a 25-mm ²monopolar electrode (model MF 25: TECA Corporation, Pleasantville, NY) with ²the distal 2 mm ²exposed. The electrode was implanted "free hand" in the DREZ with use of the ²intraoperative ²microscope to a 2mm depth. The axis of implantation was approximately 35 to 45 ²degrees ²medially and was the same axis used for DREZ microcoagulation. Ground and ²reference Grass ²subdermal electroencephalographic electrodes were placed in exposed ²paraspinous muscle ²bilaterally. Spontaneous electrophysiological recordings were obtained using ²an evoked potential ²averager (Cascade Pro 32 Channel; Cadwell Laboratories, Kennewick, WA) at a ²gain setting of ²50 with the high-frequency filter set at 3 kHz and the low-frequency filter ²set at 100 Hz. The ²recordings were one second in duration.²

[0073] Methods of data analysis were described in a previous publication and ²were used in this ²study. (Falci S, Best L, Bayles R, Lammertse D, Starnes C. J Neurosurg Spine ²97:193-2000, ²2002) In brief, the initial data were analyzed by RMS (expressed in ²microvolts), frequency and ²voltage in the waveform by FFT, and area under the waveform curve (expressed ²in microvolts ²per millisecond). These same analyses were performed to test data subsequent ²to DREZ ²microcoagulation, Both RMS analysis and²13²Date Recue / Date Received 2022-04-14²<DP=14>²WO 2018 / 071893 PCT / US2017 / 056745²area-under-the-waveform data provided a single numerical value of the recorded ²neuro-electrical ²energy. Analyses were performed using a subroutine in the Cadwell Cascade ²software. A ²phenomenon that we describe as "spindles' was examined by passing the initial ²data through a ²tight digital filter with a band pass of 65 to 100 Hz. A visual count was made ²of the number of ²spindle bursts in the 1-second recording, excluding artifacts caused by ²cardiac electrical activity ²or electrode movement. With these analyses, two distinct electrophysiological ²DREZ activities ²were found, consistent with our previous study. Those activities showing lower ²voltage and ²frequencies, smaller area under the waveform curve, and fewer than three ²spindles were ²considered to be non-pain-producing DREZ activity (Figure 3) those showing ²higher voltage and ²frequencies, greater area under the waveform curve, and more than 3 spindles ²were considered ²to be regions of abnormal pain-producing neuro-electrical hyperactivity ²(Figure 4). Analyses were ²also performed subsequent to DREZ microcoagulation of regions of abnormal ²neuro-electrical ²hyperactivity. It is notable that values of diminished activity are even less ²than those consistent ²with non-pain-producing activity (Figure 5).²

[0074] DREZ microcoagulation was performed using a needle tipped ²electrocautery (Covidien ²Force FX electrosurgical generator, Valleylab, Inc, Boulder, CO) at a setting ²of 10 desiccate for a ²one second pulse, with 1 mm of separation in all DREZs in which spontaneous ²neuro-electrical ²hyperactivity was demonstrated. Following microcoagulation, neuro-electrical ²hyperactivity was ²again measured. If recorded traces showed absence of neuro-electrical ²hyperactivity, no further ²lesions were made. If however, recorded traces continued to show spontaneous ²neuro-electrical ²hyperactivity, microlesioning was repeated.²

[0075] Complete or near complete relief of all below-level neuropathic pains ²were achieved in all ²three patients. 100% pain relief was reported in gluteal, rectal, genitalia, ²upper leg, and lower leg ²and feet in all three patients, with the exception of residual burning and ²electrical sensations in the ²feet in one patient, rated 0-3 out of 10 in intensity, and a non-painful ²"tingling" and "warmth" ²sensation in the feet of another rated 0-3 out of 10 in intensity (Figure 7). ²Two of the three patients ²were able to completely wean off of all their preoperative pain medications ²which included ²DuragesicTm patch, NeurontinTm, and CymbaltaTm. Follow-up examination was ²performed 1 1 / 2, 2 ²1 / 2 and 11 years respectively in the three patients. Follow-up observation ²regarding pain relief ²was accomplished by telephone interview and outpatient evaluation. Pain ²evaluation was a ²verbal scale in which scores of 1-10 indicated intensity of pain.²

[0076] All three patients underwent preoperative and postoperative ASIA ²sensory and motor ²testing. There were no changes in testing as anticipated in these ASIA A ²patients with surgery ²only caudal to the injury site. There were no cases of wound infection, deep ²venous thrombosis,²14²Date Recue / Date Received 2022-11-22²<DP=15>²CA 03040482 2019-04-12²WO 2018 / 071893 PCT / US2017 / 056745²pulmonary embolus, cerebrospinal fluid leakage, or death. One patient ²developed a postoperative ²pseudomeningocele which resolved non-surgically within six months ²postoperatively. At 10 ²months postoperatively, the same patient developed urosepsis with disc space ²infection and ²instability at L1 / 2 requiring surgical stabilization. At 3 years ²postoperatively, the same patient ²developed urosepsis with epidural abscess formation and instability at T12 / L1, ²once again ²requiring stabilization. These episodes of instability were not considered ²treatment-related. ²Reported pain reduction remained subsequent to these two surgeries.²

[0077] DREZ hyperactivity in T3-T7 spinal cord regions will result in truncal ²below-level central ²pain, hyperactivity in T8-T10 spinal cord regions, gluteal, rectal, and / or ²genitalia region ²below-level pains, and hyperactivity in T11-L1 DREZs upper, lower leg, and ²foot below-level pain. ²All three patients in this study experienced upper leg, lower leg, and foot ²below-level pains. All ²three patients additionally had DREZ hyperactivity recorded in more ²traditional DREZs of sensory ²rootlets subtending classical somatic pain for these same body regions (i.e. ²L1-S1) (Table 2).²Table 2²Patient 1 2 3²Levels of operative DREZ hyperactivity²T10 X X²T11 X X X²T12 X X X²L1 XXX²L2 X X X²L3 X X X²L4 XXX²L5 X , X X²S1 XXX²52 X X X²

[0078] Two of the three patients experienced below-level gluteal, rectal, and ²genitalia region ²pains and both had DREZ hyperactivity recorded in 110 DREZs. Both additionally ²had DREZ ²electrical hyperactivity recorded in the more traditional DREZ of sensory ²rootlets subtending ²classical somatic pain from these regions (i.e. 32) (Table 2).²

[0079] Spinal cord caudal to the level of a completely transected spinal cord ²(the neurological ²injury) can be a source of below-level spinal cord injury neuropathic pain, ²and with regard to the ²patients in this example, essentially a sole source. Further, below-level SCI ²neuropathic pain ²transmission can be substantially through the sympathetic chain and SNS-²mediated pain ²pathways. To reach supraspinal pain centers, a new neuronal circuit must form ²post-injury,²<DP=16>²WO 2018 / 071893 PCT / US2017 / 056745²perhaps through reactive C-fiber sprouting, between hyperactive DREZs caudal ²to the level of ²spinal cord transection, and the sympathetic chain and spinal cord regions ²more cephalad to the ²transection, and / or the brain directly through the sympathetic chain and then ²occipital foramen. ²Transmission of DREZ hyperactivity through this new circuit may occur through ²ventral roots to ²the sympathetic chain, or in a retrograde fashion through dorsal roots to the ²sympathetic chain, ²with re-entry into the spinal cord by way of ventral or dorsal roots (Figure ²8). At-level somatic ²C-fiber afferents coursing from the inguinal region and below-level ²sympathetic C-fiber afferents ²coursing from the foot are shown converging on the same L1 DREZ. The proposed ²second-order ²sympathetic afferent pathways are similar to sympathetic efferent pathways. ²Transection of the ²spinal cord cephalad to the L1 level could arrest at-level pain transmission ²to the supraspinal ²centers, but not below-level pain transmission. Additional support of this ²proposed mechanism is ²the historical failure of cordectomy and cordotomy to relieve below-level SCI ²neuropathic pain, as ²well as our own personal experience with cordectomy.²Example 4²

[0080] Figure 9 shows normal electrical activity of the spinal cord DREZ of ²T10 in regions below ²the cervical level of SCI in a complete quadriplegic with leg pain. Figure 10 ²shows electrical ²hyperactivity in the DREZ of T11 below the cervical level of complete spinal ²cord injury ²corresponding to a somatotopic map of leg DREZ pain generating tissue in cord ²regions. That the ²DREZ pain generator is located caudal to the cervical level of complete SCI ²and that SCI ²neuropathic pain is perceived below the level of complete SCI shows that the ²central neuropathic ²pain is sympathetically mediated. The presence of pain perceived by the ²patient to be below the ²level of spinal cord injury shows that pain is below the level of ²sympathetically mediated pain. ²

[0081] Having described several embodiments, it will be recognized by those ²skilled in the art that ²various modifications, alternative constructions, and equivalents may be used ²without departing ²from the spirit of the disclosure. Additionally, a number of well-known ²processes and elements ²have not been described in order to avoid unnecessarily obscuring the ²disclosure. Accordingly, ²the above description should not be taken as limiting the scope of the ²disclosure.²

[0082] Those skilled in the art will appreciate that the disclosed embodiments ²teach by way of ²example and not by limitation. Therefore, the matter contained in the above ²description or shown ²in the accompanying drawings should be interpreted as illustrative and not in ²a limiting sense.²16²Date Recue / Date Received 2022-04-14²

Claims

<DP=1>²What is Claimed is:²1. Use of 2-(2-oxo-4-propylpyrrolidin-1-yl)butanamide for treating ²neuropathic pain in a ²spinal cord injured individual in need thereof, wherein the neuropathic pain ²is perceived by the ²individual to be below the level of spinal cord injury.²2. The use according to claim 1, wherein the pain is generated by spinal ²cord tissue caudal ²to the level of the spinal cord injury.²3. The use according to claim 1 or 2, wherein the 2-(2-oxo-4-²propylpyrrolidin-1-²yl)butanamide is for use intrathecally below the level of the spinal cord ²injury.²4. The use according to any one of claims 1 to 3, wherein the neuropathic ²pain is ²sympathetically mediated.²17²Date Recue / Date Received 2023-07-10²