EP3969913A1 - Biomarker for assessment of spinal cord injury - Google Patents

Abstract

The present application relates to biomarkers for spinal cord injury. The inventors have shown that calpain-specific sodium channel cleavage fragments represent a sensitive and accurate biomarker for spinal cord injury (SCI) and spasticity. As shown herein, both kinetics and stability over time of calpain-generated sodium channel fragments support their use as biomarker to identify mild to severe SCI. The present invention therefore relates to the use of such biomarkers for assessing the presence and/or the severity of the injury in the acute phase of SCI; monitoring SCI or spasticity progression; predicting motor outcomes; selecting the best course of treatment; and/or evaluating the efficacy of therapies for SCI and notably for spasticity, notably therapies targeting calpain(s).

Description

Biomarker for assessment of spinal cord injury

FIELD OF THE INVENTION

The present invention provides biomarkers for assessment of spinal cord injury and methods of using such biomarkers to determine the severity of the SCI, a prognosis, and direct or monitor a treatment for a patient with a SCI.

BACKGROUND

There are about 35,000 and 259,000 patients with spinal cord injury (SCI) in France and USA, respectively (van den Berg et al., 2010). It is unclear how many people in the world are currently living with SCI, but international incidence data suggest that every year between 250 000 and 500 000 people become spinal cord injured.

SCI is a physically devastating condition that primarily affects younger populations and often results in life-long functional disability. At post-injury year 1 , only 1 1 % of persons with SCI are employed. SCI not only affect one’s health, but also generates a huge economic burden on the society. The estimated lifetime costs varies according to severity of SCI from 1 million $ for a paraplegic patient to more than 3 million $ for tetraplegia. The yearly health care and living expenses is estimated to be 16 billion $ in the U.S. Because road traffic crashes remain the most common cause of SCI, its healthcare burden and social costs are expected to continue to rise worldwide with the economic emergence of developing countries.

Not surprisingly, the initial questions of injured patients, families, and friends typically center on the prognosis for recovery. However, it still remains a key concern for the physician as recovery following SCI is particularly variable from individual to individual. Weeks to months after SCI, spasticity may develop in ¾ of patients with SCI (Skold, 2000). The multifold symptoms include hypertonicity (increased muscle tone), clonus (a series of rapid muscle contractions), exaggerated deep tendon reflexes (hyperreflexia), muscle spasms, scissoring (involuntary crossing of the legs), and fixed joints (contractures). The degree of spasticity may also vary from mild muscle stiffness to severe, painful, and uncontrollable muscle spasms that negatively influence quality of life and impeding rehabilitation efforts (Parziale et al., 1993; Kirshblum, 1999; Burchiel and Hsu, 2001 ). Therefore, an early, accurate diagnostic test after SCI designed to target therapeutic and rehabilitation strategies would be a highly desirable prognostic tool. Physicians are thus unanimous in insisting that at the initial damage of the spinal cord (SC), the patient should be carefully evaluated in order to help selecting an appropriate neuroprotective treatment. Yet, despite intensive research in the field, development of a reliable tool allowing assessment of patient who endured an SCI is still not available. Currently, in clinical practice, neurologic impairment after SCI remains usually assessed by neuroimaging techniques and neurological examination according to the American spinal injury association impairment scale (ASIA), but none of these diagnostic tools is sufficiently sensitive to accurately predict neurologic outcome and define the severity of the lesion.

Various proteins have demonstrated to be associated with SCI pathogeny and have thus been proposed as biomarker for SCI, such as GFAP and calcium-binding protein S100-beta (S100p), neurofilament proteins, SBDP150 and UCH-L1 , neurofilament light chain (NF-L), phosphorylated form of neurofilament heavy chain (pNF-FI), tau in biofluids (CSF and serum), inflammatory cytokines such as as IL-6, IL1 b, IL12, interferon gamma (IFNy), and TNFa. Multiple candidate protein biomarkers known to be linked to the pathology have been studied for their potential to predict neurological outcome over time. This hypothesis-driven approach, however, has yielded only minimal success, and the reported individual markers lacked sensitivity and correlation with specific outcome measures, which highlights the need for an unbiased high-throughput screening approach to identify novel candidate biomarkers (Ydens et al., 2017).

Among factors that contribute to spasticity after SCI, a change of the excitability of motoneurons (MNs) innervating muscles from hypoexcitability during spinal shock to hyperexcitability during spasticity was presumed. According to this concept, the SCI predisposes motoneurons to express self-sustained spiking activity that arises from a prolonged depolarization known as “plateau potential” and contributes to muscle spasms (Li et al., 2004). In a series of papers, the inventors demonstrated that the persistent sodium current (/_Nap) is largely expressed in the locomotor network (Tazerart et al., 2007; Tazerart et al., 2008; Brocard et al., 2010; Brocard et al., 2013) and is critical for driving plateau potentials in motoneurons (Bouhadfane et al., 2013). The inventors also showed that /_Nap is increased after SCI and has been pointed out as a major contributor of spastic reflexes in rodents with SCI (Brocard et al., 2016). The /_NHP has been also assumed to mediate spasms in patients with SCI (Heckman et al., 2008). Thus, it appears that l_Nap is upregulated after SCI and contributes to spasticity. However, the mechanisms causing the hyperexcitability of the motoneurons were not fully understood until recently.

The inventors previously identified (see Brocard et Al., 2016)) the molecular mechanism responsible for the alteration of l_Nap after SCI. They demonstrate that the up-regulation of /_Nap after SCI is accompanied by a proteolytic cleavage of the a- subunit of voltage-gated sodium channels (Nav) into breakdown products of around 120 kDa (a-Nav-BDP120’). Immunoblot revealed a proteolysis of Nav channels and biochemical assays identified calpain as the main proteolytic factor. Calpain- dependent cleavage of Nav channels following SCI was associated with an upregulation of /_Nap in motoneurons. Likewise, calpain-dependent cleavage of Nav channels caused elevation of /_N3P. Pharmacological inhibition of calpain reduced the cleavage of Nav channels, /_N3P in motoneurons and spasticity in rats with SCI. This study demonstrates that Nav channel expression in lumbar motoneurons is altered after SCI and shows a tight relationship between the calpain-dependent proteolysis of Nav channels, the upregulation of /_N3P and spasticity.

Recent studies have suggested that fragments from the calpain-mediated cleavage of a-ll spectrin were potential sensors of calpain activity. However, the a-ll spectrin protein is also a major substrate for the protease caspase-3, which is involved in cellular apoptosis after SCI. Furthermore, spectrin breakdown is transiently increased only during the acute phase of SCI, making it impossible to monitor the progression of the injury over time, or again the efficacy of novel therapies in chronic phase of the SCI. Finally, as spectrin is highly expressed in red blood cells, its diagnostic values as a SCI-specific marker can be questioned, especially in polytraumatized patients.

In sum, there is still a need in the art to identify accurate biomarkers in humans that can be used from acute staging of the patient having endured an SCI to long-term following of the chronic stage of the disease. There is also a need for a biomarker that can be rapidly detected notably in biological samples that are easily obtainable such as serum, blood or plasma sample. SUMMARY OF THE INVENTION

The present disclosure relates to the following embodiments:

The use of one or more proteolytic fragments of one or more sodium channels as a biomarker for:

- diagnosing spinal cord injury in a patient,

- assessing the clinical grade of spinal cord injury or predicting the motor outcome of a patient suffering from spinal cord injury,

- predicting the efficacy of a treatment for spinal cord injury in a patient, or for

- monitoring the motor outcome or treatment efficacy in a patient suffering from spinal cord injury; wherein said one or more proteolytic fragments are detected in sample from said patient, notably a serum, whole blood or plasma sample from said patient.

The present invention also relates to:

- an in vitro method for diagnosing spinal cord injury (SCI) in a patient comprising the detection in a sample obtained from said patient of at least one marker from proteolytic cleavage of one or more sodium channel, typically wherein an increase of said at least one marker as compared to a control, is indicative of spinal cord injury;

- a method for assessing the clinical grade of spinal cord injury or predicting motor outcome in a patient suffering from spinal cord injury or suspected to suffer from spinal cord injury comprising the detection in a sample obtained from said patient of at least one marker from proteolytic cleavage of one or more sodium channel, wherein the level of said at least one marker is indicative of the clinical grade or the motor outcome of the patient;

- a method for predicting the efficacy of a treatment for spinal cord injury in a patient comprising the detection in a sample obtained from said patient of at least one marker from proteolytic cleavage of one or more sodium channel, typically wherein a decrease of said at least one marker as compared to a control is indicative of treatment efficacy;

- a method for monitoring the motor outcome or treatment efficacy in a patient suffering from spinal cord injury comprising the detection in a sample obtained from said patient of at least one marker from proteolytic cleavage of one or more sodium channel at 2 or more time points after spinal cord injury, typically wherein a decrease of said at least one marker as compared to a control is indicative of motor improvement.

Typically, the sodium channel is selected from Nav1.1 , Nav1.2, Nav1.3, Nav1.6, Navi .7, Navi .8 and Navi .9. In one embodiment of the present disclosure, the at least one marker from proteolytic cleavage of one or more sodium channel is a calpain-specific sodium channel proteolytic fragment, typically the calpain-specific sodium channel proteolytic fragment is a 120 kDa sodium channel fragment.

In some embodiments of the present disclosure, the spinal cord injury results from traumatic injury, ischemic neuropathies or compression syndrome.

The level of sodium channel cleavage fragments can be measured by liquid chromatography, gas chromatography, mass spectrometry, radioimmunoassays, immunofluorescent assays, FRET-based assays, immunoblot, ELISAs, or liquid chromatography followed by mass spectrometry. Typically, the biological sample is selected from a spinal cord sample, cerebrospinal fluid, whole blood, plasma, serum, saliva and urine.

In some embodiments, the patient is a mammalian, notably a human and typically a pediatric patient.

Control(s) can be obtained from a biological sample from at least one healthy patient, from said patient suffering from spinal cord injury (in the absence of treatment of after having started an SCI treatment) or is/are a pre-determined reference value(s).

A further object of the present disclosure relates to a neutroprotective drug for use in a method of treatment of a patient suffering from spinal cord injury, wherein the methods comprises the determination of the clinical stage of spinal cord injury according to claim 2.

Said neuroprotective drug can be selected from methylprednisolone, 17a-estradiol, 17P-estradiol, ginsenoside, progesterone, simvastatin, deprenyl, minocycline, resveratrol, other glutamate receptor antagonists, antioxidants, calpain-related therapeutics agents and combination thereof.

Calpain-related therapeutic agents typically include ALLNal, MDL28170, AK295, Calpastatin, SNJ1945, BDA-410 ABT-957, Olesoxime, Cyclosporine A, E-64d (EST, Estate, loxistatin and rexostatine), AK-295 (CX 295), C-101 (myodur and CLA), CYLA, C201 (neurodur and CLA), CEP-3453, CEP-4143, Calpeptin (IPSI-001 ), A- 705239 (BSF 409425), A-705253, Ala-1.0, and combination thereof.

Another object of the present disclosure relates to the use of proteolytic cleavage of one or more sodium channel as a biomarker for diagnosing spinal cord injury in a patient, for the prognosis of the clinical outcome of a patient suffering from spinal cord injury, or for predicting the efficacy of a treatment for spinal cord injury, or for monitoring the motor outcome or treatment efficacy in a patient suffering from spinal cord injury.

Lastly, the present disclosure encompasses a kit for use in a method according to any one of claims 1 -1 1 , wherein the kit comprises a reagent for detecting sodium channel cleavage fragments and instructions for implementing said methods.

Other features and advantages of the invention will be apparent from the following detailed description and figures and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 : Cleavage of Nav channels correlates with the level expression increase of m-calpain after SCI. (a,c,e,g) Immunoblots of lumbar segments in sham-operated and rats with SCI probed with a calpain-l polyclonal (a), calpain-l monoclonal (b), calpain-ll (c) or Pan-Nav antibody. One rat per lane. (b,d,f,h) Quantification of bands in rats with SCI, normalized to sham-operated controls. ^**P < 0.01 , ^***p < 0.001 , comparing sham-operated to SCI; Mann-Whitney test. (i,j,k) The relative level expression of the a-Nav-BDP120’ band shown as function of the relative level expression of calpain-l probed with a polyclonal (i) or monoclonal (j) antibody in neonatal rats or probed with monoclonal antibody in adult rats (k). Each dot represents a single rat. The continuous line is the best-fit linear regression function. ^**P< 0.01 , ^*** P< 0.001 Spearman correlation test.

Figure 2: Cleavage of Nav channels in rats with severe acute SCI. (a) Pan-Nav immunoblot of lumbar segments in sham-operated and adult rats with acute SCI (within the 24 hours after SCI). One rat per lane (b) Quantification of ~250-kDa and ~120-kDa bands in rats with SCI, normalized to sham-operated controls. ^*P < 0.05, ^**P < 0.01 , comparing sham to SCI; Mann-Whitney test (c) Pan-Nav immunoblot of lumbar segments at different time after SCI (two right lines) compared to sham- operated adult rats (left line).

Figure 3: Time course profile and diagnostic accuracy of a-Nav-BDP120’ after

SCI. (a,d) Spectrin (a) and Pan-Nav (d) immunoblots of lumbar segments in sham- operated and neonatal rats with SCI at different time after injury (1 , 3 and 5 days after SCI). One rat per lane (b) Quantification of bands at different time after SCI in neonatal rats, normalized to sham-operated controls. ^*P < 0.05, comparing sham to SCI; Mann-Whitney test.

Figure 4 : Early detection of a-Nav-BDP120’ in adult rats with a mild SCI. (a,c,e)

Pan-Nav (a,c) and calpain-l (e) immunoblots of lumbar segments in sham-operated and adult rats with a mild contusion of the spinal cord (SCI) at different time after injury (1 and 2 days after SCI). One rat per lane. (b,d,f) Quantification of bands at different time after SCI in rats, normalized to sham-operated controls. ^*P < 0.05, comparing sham to SCI; Mann-Whitney test. The relative level expression of the a- Nav-BDP120’ band shown as function of the relative level expression of calpain-l in adult rats (g). Each dot represents a single rat. The continuous line is the best-fit linear regression function. ^*** P< 0.001 Spearman correlation test.

Figure 5 : Detection of a-Nav-BDP from Nav channels in adult rats with SCI. (a)

Navi .6 immunoblot of lumbar segments in sham-operated and adult rats with chronic SCI. (b) Quantification of immunoblots (from plasma samples of adult rats) bands at different time before (TO) and after SCI in rats, normalized to the TO value. ^*P < 0.05, comparing TO to SCI; Mann-Whitney test. (c,d) Quantification of Pan-Nav immunoblots (from plasma samples of adult rats) bands at different times before (TO) and after SCI in rats, normalized to to sham-operated controls. ^*P < 0.05, comparing sham to SCI; Mann-Whitney test. (b,d) corresponds to the sum of all a-Nav-BDP values (a) are only a-Nav-BDP 120’ values.

Figure 6: Promising therapies targeting calpains prevent the SCI-induced increase of a-Nav-BDP120’ in rats, (a) Pan-Nav immunoblot in adult rats with SCI acutely treated with vehicle or MDL28170 (10 mg/kg/d for 2 days beginning within the hour after SCI). One rat per lane (b) Quantification of immunoreactive ~250-kDa and ~120-kDa bands in sham-operated (white) and MDL28170-treated (grey) rats with SCI, normalized to vehicle-treated rats with SCI (black). ^*P < 0.05, comparing sham- operated to vehicle-treated rats; One-way ANOVA. (c) Pan-Nav immunoblot in adult rats with chronic SCI (60 d after SCI) treated with demeclocycline (DMC; 45 mg/kg every 12 h for 5 days). One rat per lane (d) Quantification of immunoreactive ~250- kDa and ~120-kDa bands in DMC-treated rats (black) with SCI, normalized to vehicle-treated rats with SCI (white); comparing sham-operated to vehicle-treated rats; Mann-Whitney test (e) Pan-Nav immunoblot in neonatal rats with SCI treated with vehicle or the 5-HT₂ receptors agonist DOI. One rat per lane (f) Quantification of immunoreactive ~250-kDa and ~120-kDa bands in sham-operated (white) and DOI- treated (grey) rats with SCI, normalized to vehicle-treated rats with SCI (black). ^*P < 0.05, comparing sham-operated to vehicle-treated rats; One-way ANOVA.

DETAILED DESCRIPTION

The following description outlines the invention summarized above. The disclosure, however, is not limited to the particular methodology; protocols, or reagents described and as such may vary. Likewise, the terminology used herein describes particular embodiments only, and is not intended to limit the scope of the invention.

Calpains are known to be recruited (see Banik et al., 1997; Du et al., 1999) and to play an important role in the development of spasticity after SCI (see Brocard et al., 2016 ) .

The inventors have now discovered that detection of sodium channel cleavage fragment(s), and more particularly calpain-specific sodium channel cleavage fragments represent a sensitive and accurate biomarker for spinal cord injury (SCI) and spasticity. The results provided therein support their use as biomarker to identify mild to severe SCI. The results also demonstrate that both kinetics and stability over time of calpain-generated sodium channel fragments allow the use of said sodium channel cleavage fragment for:

(i) assessing the presence and/or the severity of the injury in the acute phase of SCI;

(ii) monitoring SCI or spasticity progression;

(iii) predicting motor outcomes;

(iv) selecting the best course of treatment; and/or

(v) evaluating the efficacy of therapies for SCI and notably for spasticity, notably therapies targeting calpain(s).

The inventors have further provided evidence that detection of sodium channel breakdown products (BDP) can be acutely detected in the plasma of patient suffering from SCI and that said sodium channel BDP chronically persists in said biological sample of said patients.

Thus, the present disclosure is notably based on the characterization of calpain activation by means of detection sodium channel breakdown products (BDP), for example the a-Nav-BDP120’ measurements that represent potential sensitive biomarkers of the SCI and/or spasticity in human patients. Accordingly, the invention provides methods, and kits for detecting and quantifying the level of sodium channel breakdown products, notably a-Nav-BDP120’ from at least one Nav channels in a biological sample obtained from the patient, wherein the measured level of sodium channel breakdown products is indicative of the presence of SCI, the seriousness of the injury and helps direct the most appropriate treatment.

Definitions

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the relevant art. For convenience however, the meaning of certain terms and phrases employed in the specification, examples, and appended claims are provided below.

The singular forms "a," "an," and "the" include plural reference unless the context clearly dictates otherwise.

Calpains are a family of ubiquitously expressed intracellular calcium-activated cysteine proteases. Humans have 15 genes that encode calpain catalytic subunits, which are now called CAPNn (n=1 , 2, 3, and 5~16), and their gene products are correspondingly called CAPNn (n=1 , 2, 3, and 5~16). There are two genes, CAPNS1 and CAPNS2, for calpain small regulatory subunit, and one, CAST, for calpastatin. m-Calpain and m-calpain are the two major calpains ubiquitously found in mammalian cells m- and m-calpains are activated in vitro by micromolar and millimolar Ca²⁺ concentrations, respectively. (These molecules were also called calpain I and calpain II, previously.) m- and m-calpains are heterodimers consisting of a common, smaller calpain regulatory subunit (CAPNS1 , also called 30 K) and a distinct, larger catalytic calpain subunit (CAPN1 or CAPN2, also called pCL and mCL, respectively), which are ca. 60% identical in their protein sequences. To avoid confusion, these enzymes are now proposed to be called by their subunit composition, CAPN1/S1 and CAPN2/S1 (see Handbook of Proteolytic Enzymes, Vol. 2, 2013, Pages 1995-2011 )

A“biological sample" as herein intended encompasses a variety of sample types obtained from an organism that may be used in a diagnostic, prognostic or monitoring assay or method as herein disclosed. The term encompasses any liquid samples of biological origin such as cerebrospinal fluid (CSF), urine, saliva, whole blood, plasma, serum, sputum, semen, faeces, a nasal swab, tears, solid tissue samples, such as a biopsy specimen, or tissue cultures or cells derived there from and the progeny thereof. The term also encompasses samples that have been manipulated in any way after procurement, such as treatment with reagents, solubilization, or enrichment for certain components.

"Diagnosis" and "diagnosing" generally includes a determination of a subject’s susceptibility to a disease or disorder, a determination as to whether a subject is presently affected by a disease or disorder, a prognosis of a subject affected by a disease or disorder ( e.g ., identification of motor outcomes of patient suffering of SCI), and therametrics ( e.g ., monitoring a subject’s condition to provide information as to the effect or efficacy of therapy).

The terms " prognosis " and " prognose " refer to the act or art of foretelling the course of a disease. Additionally, the terms refer to the prospect of evolution {e.g. motor outcomes) and recovery from a disease as anticipated from the usual course of that disease or indicated by special features of the individual case.

" Individual ," "subject," and "patient," used interchangeably herein, refer to any mammalian subject for whom diagnosis, prognosis, monitoring, treatment, or therapy is desired. In one embodiment, the individual, subject, host, or patient is a human and notably a pediatric patient. Pediatric patients include newborn, infants, children and adolescents. The methods of the invention find use in experimental animals, in veterinary application, and in the development of animal models for SCI, thus other subjects may include, but are not limited to, cattle, horses, dogs, cats, guinea pigs, rabbits, rats, primates, woodchucks, ducks, and mice.The terms "treatment," "treating," "treat," and the like refer to obtaining a beneficial or desired result including pharmacological and/or clinical result. The effect may be prophylactic in terms of completely or partially preventing a disease or symptom thereof and/or may be therapeutic in terms alleviation or amelioration of one or more symptoms or conditions, diminishment of extent of disease, stabilized (i.e. not worsening) state of disease, preventing spread of disease, delay or slowing of disease progression, reversal of disease, amelioration or palliation of the disease state, and remission (whether partial or total). "Treatment" thus covers any treatment of a disease in a mammal, particularly a human, and includes: (a) preventing the disease or symptom from occurring in a subject which may be predisposed to the disease or symptom but has not yet been diagnosed as having it; (b) inhibiting the disease symptom, i.e., arresting its development; or (c) relieving the disease symptom, i.e., causing regression of the disease or symptom.

As used herein, the term“decrease” or“increase” means a statistically significant decrease or increase of a value, as compared to a control, or reference value, preferably, at least 10; 20; 30; 40; 50; 60; 70; 80; or 90%, decrease or increase of the control or reference value. The expression “detection” or“detecting” a marker as used herein encompasses qualitative and qualitative detection. By qualitative detection it is intended that the presence, or absence (i.e., non-detectable signal as per the used detection method), of the marker is assessed. By quantitative detection is it herein intended that the level or concentration of the marker is measured.

Spinal cord injury and marker(s) from proteolytic cleavage of Nach

“Spinal cord injury’ (herein referred as SCI) is used herein in the broadest sense and indicates any functional or structural spinal injury (including typically a bruise, also called a contusion; a partial tear, or a complete tear (called a transection) in the spinal cord) that may result from traumatic injury, ischemic neuropathies, or from a compression syndrome.

Typically, a spinal cord injury as herein intended involves a motoneuron injury or damage. Examples of“ischemic injuries or neuropathies”, as used in the context of the present disclosure include any cardiovascular accident and/or spinal cord ischemia.

Examples of “traumatic injuries”, as used in the context of the present invention include spinal cord injury or peripheral nerve injury.

Examples of “compression syndromes”, as used in the context of the present invention include spinal or nerve compressions, such as disc herniation-induced nerve compression, typically disc herniation-induced spinal nerve compression.

In some embodiments, the spinal cord injury can also be induced by a number of risk factors including, but not limited to exposure to cytotoxic compounds, radiation exposure, viral infections, alcohol and drug abuse, autoimmune or neurodegenerative diseases.

Definitions and clinical syndromes that are typically associated with SCI arising from direct cord compression, haemorrhage, traction forces, or any other space-occupying lesion, have been agreed by the International Standards for Neurological and Functional Classification of SCI. ASIA, American Spinal Injury Association. The ASIA impairment scale (modified from the Frankel Classification) is an internationally recognized scale to classify the clinical extent of SCI. Impairment is classified from A-E, the estimated incidence of each type of injury is indicated in brackets (see for reference Roberts TT et al, 2016; or Krishna V et al., 2014).

A=Complete:No sensory or motor function is preserved in the sacral segments S4- S5 (45%)

B=lncomplete: Preservation of sensory but not motor function below the neurological level and includes the sacral segments S4-S5 (15%)

C=lncomplete: Preservation of motor function below the neurological level. More than half of key muscles below the neurological level have a muscle grade of <3 (10%)

D=lncomplete: Preservation of motor function below the neurological level. More than half of key muscles below the neurological level have a muscle grade of >3 (30%)

E=Normal: Sensory and motor function is normal

In one embodiment of the present disclosure, a subject according to the present disclosure is suffering from acute spinal cord injury. Acute spinal cord injury typically refers to a period of less than 1 month, preferably less than 3 weeks, less than 2 weeks, less than 1 week, less than 4 days, less than 48 hours, less than 24 hours, less than 12 hours, less than 6 hours, less than 2 hours or less than 1 hour after the injury or damage.

In one other embodiment of the present disclosure, a subject of the present invention is suffering from chronic spinal cord injury. Typically, chronic spinal cord injury refers to a permanent and/or progressive interruption in the conduction of impulses across the neurons and tracts of the spinal cord. It may be due to mechanical distortion or vascular ischemia of the spinal cord arising from trauma, tumor, infection, other space-occupying lesions, or any of the risk factors as above mentioned. The term is generally used when elements of spinal cord injury have been present for at least 6 months, notably at least 1 year. Commensurate neurologic deficits occur that may be stable or progressive and lead to disability with spasticity, joint contractures, sensory changes, and sphincter and locomotion abnormalities. In another embodiment of the present disclosure, a subject of the present invention may also be at risk of suffering of spinal cord injury (SCI). For example such subject may have suffered from traumatic injury, ischemic neuropathies, or from a compression syndrome. In some cases a subject of the present invention has been exposed to cytotoxic compounds, radiation exposure, viral infections, autoimmune diseases, neurodegenerative diseases, or has alcohol or drug abuse history.

In some embodiments of the present invention, the spinal cord injury results from traumatic injuries, compression syndromes or any space-occupying lesion as defined above. Typically, the spinal cord injury results from traumatic injury.

Sodium channels” or“Nav channels” or“Nach” as depicted herein are voltage-gated sodium channels. Mammalian Nav channels are formed by a large pseudotetrameric pore-forming a subunit (of approximately 260 kDa) organized in four homologous domains, which each contains six transmembrane segments that associates with one or two b subunits (30-40 kDa) (see for review Catterall, 2012). By“around” or “approximately” 260 kDa it is herein intended that the molecular weight of the proteolytic fragment is of 260 kDa with an accepted variation of ± 25 %, notably ± 20 %, more specifically ± 15%, ± 10% and typically ± 5%. Such variation may depend on both the a-subunit and/or on the method used to detect said protein fragment.

To date, nine a-subunits (Navi 1 -Nav1.9) have been functionally characterized. With the exception of the Navi .4 and Navi .5 channels, which are mainly found in skeletal and cardiac muscle respectively, the others are strongly expressed in neurons, especially those in the spinal cord.

Thus, in some embodiments of the present disclosure, the expression“at least one sodium channel’ refers to at least one Nav subtype that include Nav1.1 , Nav1.2, Nav1.3, Nav1.6, Nav1.7, Nav1.8 and Nav1.9 channels. In some embodiments, the sodium channel is selected from the group comprising Nav1.1 , Nav1.6, Nav 1.7, Navi .8 and Navi .9, notably Navi .1 , Nav 1.3; Navi .6, Nav 1.7, Nav 1.8 and Navi .9, more specifically from the group comprising, Nav1.1 , Nav1.6; Nav1.8; Nav1.9 and even more specifically from the group comprising Nav 1.1 and Navi .6.

As previously mentioned, the present disclosure is based on the detection, in a biological sample, of at least one marker from proteolytic cleavage of one or more sodium channel (Nach). Typically, the term sodium channel refers to the a-subunit of the sodium channel. Thus, the terms “marker(s) from proteolytic cleavage”, or “breakdown product(s)”, or“proteolytic fragments )” are used interchangeably in the present disclosure and refer to fragment of the sodium channel obtained after proteolytic cleavage of the a-subunit. Said marker is used typically, according to the present disclosure, as a biomarker for diagnosis, prognosis and monitoring of spinal cord injury and spasticity.

The a-subunit has been shown to contains several sites of calpain proteolysis (see Von Reyn et al., 2009; and Brocard et al., 2016), thus typically, the marker from proteolytic cleavage of sodium channel as herein disclosed is a calpain-specific a- subunit sodium channel proteolytic fragment. In some embodiment putative calpain- proteolytic cleavage sites may be determined using the epestfind online software (http://emboss.bioinformatics.nl/cgi-bin/emboss/epestfind).

One type or a combination of 2 or more different calpain-specific sodium channel proteolytic fragments may be used as biomarker according to the present disclosure.

Typically, by“fragment” it is herein intended a part of the full a-subunit. In some embodiments a a-subunit sodium channel proteolytic fragment has a molecular weight corresponding to 80 % of less of the molecular weight of said full a-subunit, notably 75 % or less, 70% or less, 65 % or less, 60 % or less, 55 % or less or 50% or less.

In some embodiment, said calpain-specific sodium channel proteolytic fragment is obtained by cleavage of the a-subunit in the ll-lll linker region.

Said fragment can therefore encompass the lll-IV loop and the C-terminal domain (if not cleaved). Said cleavage fragment may be detected by a pan (a-subunit lll-IV loop) antibody while typically not being detected by an antibody targeting the a- subunit l-ll loop. The molecular weight of such Nach proteolytic fragment is typically of approximately 120 kDa. Indeed, in a western blotting assay for example, said Nach proteolytic fragment is typically retrieved as a protein fragment of around 120 kDa (also shortly named hereinafter“120 kDa BDP”,“a-Nach BDP120” or“Nach 120 kDa BDP”). Alternatively, said fragment can encompass the l-ll loop and potentially the N terminal domain (if not cleaved). Said cleavage fragment may be detected by a pan (a-subunit l-ll loop) antibody while typically not being detected by an antibody targeting the a-subunit lll-IV loop. The molecular weight of such Nach proteolytic fragment is typically of approximately 140 kDa. Indeed, in a western blotting assay for example, said Nach proteolytic fragment is typically retrieved as a protein fragment of around 140 kDa.

Briefly proteins are isolated from the biological sample according to known technique of the field, fractioned by electrophoresis in denaturing conditions (typically SDS- PAGE method) and probed by an antibody directed against the protein fragment (typically directed against the lll-IV loop of the a-subunit) (see also the Material & Methods section in the Examples relative to western Blot assay).

As a matter of illustration, by “around” or “approximately” 120 kDa it is herein intended that the molecular weight of the proteolytic fragment is of 120 kDa with an accepted variation of ± 25 %, notably ± 20 %, more specifically ± 15%, ± 10% and notably ± 5%. Such variation may depend on both the a-subunit and/or on the method used to detect said protein fragment. Similarly, by approximatively 140 kDa it is herein intended that the molecular weight of the proteolytic fragment is of 120 kDa with an accepted variation of ± 25 %, notably ± 20 %, more specifically ± 15%, ± 10% and notably ± 5%. Such variation may depend on both the a-subunit and/or on the method used to detect said protein fragment.

Nach BDPs and more specifically, the Nach 120 kDa BDP, the Nav 140 kDA BDP and/or any other calpain-specific sodium channel proteolytic fragment can be measured in a biological sample by various methods known to those skilled in the art. For instance, proteins can be measured by methods including, but not limited to, liquid chromatography, gas chromatography, mass spectrometry, radioimmunoassays, immunofluorescent assays, FRET-based assays, immunoblot (typically western blot), ELISAs, or liquid chromatography followed by mass spectrometry. One of skill in the art can ascertain other suitable methods for measuring and quantifying any particular biomarker protein of the invention. Western blotting is a classical method in the field and may be easily implemented in a method according to the present disclosure for the detection and assessment of Nach BDPs, notably for assessment of the a-Nav-BDP120’ from Nav channels. Briefly proteins are isolated from the biological sample according to known technique of the field, fractioned by electrophoresis in denaturing conditions (typically SDS-PAGE method) and probed by an antibody directed against the protein fragment (typically directed against the lll-IV loop of the a-subunit) (see also the Material & Methods section in the Examples relative to western Blot assay). It is to be noted that preferentially the biological samples are preserved and treated in the presence of anti-proteases (such as the Complete EDTA free Protease inhibitor Tablet from Merck) that are able to mostly inhibit cysteine and serine protease that are naturally present in the sample.

Uses and Methods of the invention

The uses and methods as herein disclosed are typically performed in vitro on a biological sample obtained from a subject.

The uses and methods of the present disclosure comprise the detection of at least one marker from proteolytic cleavage of one or more sodium channels as above defined.

For the avoidance of doubt, it will be understood that the expression“ at least one Nav channel" means that the method in question, whether carried out for a diagnostic, prognostic, monitoring or therapeutic purpose, can be carried out with any one of the listed Nav channel or a combination of two or more thereof. In other word marker(s) from proteolytic cleavage of one or a combination of sodium channels as previously defined may be detected.

Markers from proteolytic cleavage of sodium channels as herein intended have been previously defined. Typically, said markers are calpain-specific proteolytic cleavage fragments. As already mentioned, the Nach 120 kDa BDP, and/or the Nach 140 kDa BDP are representative markers, but any further Nach proteolytic cleavage fragment and more particularly calpain-specific Nach proteolytic cleavage fragment may be detected alternatively, or in combination.

Detection of a combination of Nach proteolytic cleavage fragment, more specifically of more than one Nach may improve sensitivity and/or specificity of the methods of the present disclosure.

Control(s) used for comparison purpose in the methods as herein disclosed can be obtained from a biological sample from at least one healthy patient. In other embodiment, control(s) can be obtained from at least one patient suffering from spinal cord injury, or from the tested (diagnosed, prognosed, or monitored patient) at an earlier time point (notably before and/or during treatment). In other embodiment, control(s) or is/are a pre-determined reference value(s). As used herein, " pre determined reference value " can reflect the level or concentration of the at least one marker as previously defined (and notably of a-Nach BDP120) from one or more Nav channels from a population of control subjects or from a population of patients with known levels of SCI.

In some embodiments, the present disclosure relates to the use of one or more proteolytic fragments of one or more sodium channels as a biomarker for diagnosing spinal cord injury in a patient, for assessing the clinical grade of spinal cord injury or predicting the motor outcome of a patient suffering from spinal cord injury, for predicting the efficacy of a treatment for spinal cord injury in a patient, or for monitoring the motor outcome or treatment efficacy in a patient suffering from spinal cord injury; wherein said one or more proteolytic fragments are detected in a biological sample from said patient, notably a serum, whole blood or plasma sample from said patient.

The present disclosure also relates to

- a method for diagnosing spinal cord injury (SCI) in a patient

- a method for assessing the clinical grade of spinal cord injury or predicting the motor outcome of a patient suffering from spinal cord injury,

- a method for predicting the efficacy of a treatment for spinal cord injury in a patient, or

- a method for monitoring the motor outcome or treatment efficacy in a patient suffering from spinal cord injury; said methods comprise the detection in sample obtained from said patient of at least one marker from proteolytic cleavage of one or more sodium channel, wherein typically the biological sample can be selected from a serum, whole blood or plasma sample from said patient.

Typically in such embodiment, the level of the at least one marker from one or more Na channel can be compared to control value(s) obtained from at least one healthy patient or consisting in pre-determined reference value(s) obtained from control subjects.

In other embodiments, the present disclosure relates to a method for assessing the clinical grade of spinal cord injury in a patient suffering from spinal cord injury, or suspected of having spinal cord injury, comprising the detection in sample obtained from said patient of at least one marker from proteolytic cleavage of one or more sodium channel(s), wherein the level of said at least one marker is indicative of the clinical grade of the patient.

Such method, or use of the biomarker, for assessing the clinical grade of spinal cord injury, allows to quickly stratify patients according to the severity of the SCI. The level of the at least one marker from one or more Nav channels as previously defined, and notably the level of a-Nach-BDP120 from one or more Nav channels, in the SCI patient sample, relative to level(s) in a control sample taken from at least one healthy subject or from pre-determined reference value(s), is indicative of the severity of the injury.

Typically, the level(s) of said at least one marker (typically a-Nach-BDP120) from one or more Nav channel can be measured in a sample obtained from the subject shortly after SCI (i.e., typically less than a day after injury, notably 0 to 6 hours, 0 to 4 hours and more specifically less than one hour after injury), and compare to levels in control samples taken from at least one healthy subject or to pre-determined reference values as previously defined.

The one or more proteolytic fragments use as biomarker(s) in the methods as herein described can be detected in a sample obtained from the patient at various time point after the putative spinal cord injury. By“putative spinal cord injury”, it is herein intended the injury that is suspected to be a spinal cord injury before clinical confirmation.

In some embodiments, the one or more proteolytic fragments are detected in the sample from said patient no more than 12 hours, notably no more than 10 hours, no more than 9 hours, no more than 9 hours, no more 8 hours, no more than 7 hours or no more than 6 hours after the putative spinal cord injury. For example, in some embodiments, the one or more proteolytic fragments are detected between 1 and 12 hours after the injury, notably between 1 and 10 hours, between 1 and 8 hours, between 1 and 6 hours, between 2 and 12 hours, 2 and 10 hours, 2 and 8 hours, between 2 and 6 hours. More specifically, in some embodiments, the one or more proteolytic fragments are detected between 3 and 12 hours, 3 and 10 hours, 3 and 8 hours, between 3 and 6 hours, between 4 and 12 hours between 4 and 10 hours, 4 and 8 hours, between 4 and 6 hours after the injury.

In other embodiments, the one or more proteolytic fragments are detected in the sample from said patient at least 12 hours after the putative spinal cord injury, notably at least 18h after the injury, 24 hours after the injury, 36 hours after the injury, 48 hours after the injury, 72 hours after the injury, 144 hours after the injury. In some embodiments the one or more proteolytic fragments are detected in the sample from said patient at least 1 week, notably at least 2 weeks, 3 weeks, 4 weeks, 6 weeks, after the injury. In some embodiments, the one or more proteolytic fragments are detected at least 1 month, notably at least 2 months, 3 months, 4 months, 5 months or 6 months after the injury. In some of these embodiments, the one or more proteolytic fragments are detected no more than 4 years, notably 3 years, 2 years and typically no more than 1 year after the spinal cord injury.

Typically, an increase of said marker(s) as compared to a control, is indicative of spinal cord injury.

Typically also, a decrease in the level of said marker(s) in a sample obtained from a patient (typically after starting a treatment for SCI) as compared to a control (typically the level measured in a sample obtained from said patient before starting an SCI treatment, or the level from a reference value), is indicative of treatment efficacy.

In some aspects of the present disclosure, the level of said at least one marker may be determined at multiple time points during the time course of the SCI (e.g., hours, days, weeks or months after the SCI) to determine the progression of the injury ( i.e to monitor the progression of the SCI) over the time. In such aspect the measured marker level is compared to previously determined marker levels for the same patient. Typically, an increase in the level of the at least one marker (notably of the level of a-Nach BDP120) from one or more Nav channels relative to a pre-determ ined reference value or to a previously determined marker level for the same patient is indicative of a more severe SCI. Conversely, a decrease in the level of the at least one marker (notably of the level of a-Nach BDP120) from one or more Nav channels relative to a pre-determined reference value or to a previously determined marker level for the same patient is indicative of a less severe SCI.

The present disclosure further encompasses a method for predicting, in a patient with SCI, the severity of long-term outcomes related to motor disorders such as, but not limited to, spasticity. The severity of spasticity was clearly reported or could be assessed by the Ashworth scale (Ashworth values 1-1 +, 2, and 3-4 for mild, mild, and severe spasticity, respectively). The Ashworth scale allows objective assessment of a patient's recovery following SCI and comprised of scores ranging from 0 to 4 with the following descriptions:

The method comprises the detection in sample obtained from said patient of at least one marker from proteolytic cleavage (typically the a- Nach BDP120 level) of one or more sodium channel(s), wherein the level of at said at least one marker is predictive of motor deficits. In particular, an elevated level of at least one marker as previously defined (notably of a-Nach BDP120) of at least one Nav channel relative to a pre determined reference value or to a control value is indicative of a poorer prognosis. For example, elevated a-Nach BDP120 levels are predictive of the patient having an Ashworth score of 3 to 4 upon follow-up assessment (i.e., a patient having a severe spasticity). A reduced a-Nach BDP120 level of at least one Nav channel relative to a pre-determined reference value or control value is predictive of the patient having an Ashworth score of 1 to 2 upon follow-up assessment (i.e., a patient having a mid to mild spasticity). Combination of the detection of at least one marker (typically a-Nach BDP120) from different Nav channels may improve assessment of SCI severity. Typically the patient sample is obtained shortly after SCI, for example within a week.

The present disclosure also encompasses a method for monitoring the motor outcome in a patient suffering from spinal cord injury comprising the detection in sample obtained from said patient of at least one marker from proteolytic cleavage of one or more sodium channel at 2 or more time points after spinal cord injury, wherein a decrease of said at least one marker as compared to a control is indicative of motor improvement. In such aspect the measured marker level is typically compared to previously determined marker levels for the same patient.

Also provided is a method for predicting the efficacy of a treatment for spinal cord injury in a patient having SCI comprising the detection in sample obtained from said patient of at least one marker from proteolytic cleavage of one or more sodium channel, wherein a decrease of said at least one marker as compared to a control is indicative of treatment efficacy and an increase (or even stability) of said at least one marker as compared to the control indicates a lack of therapeutic effect of said treatment.

The method can be expended to a method for monitoring the treatment/rehabilitation efficacy. Accordingly, such method further comprises measuring the level of at least one marker as previously defined (typically a-Nach BDP120) from at least one Nav channels in a biological sample obtained from the patient following neuroprotective treatment, and identifying patients responding positively to the neuroprotective treatment. A reduction in the level, abundance, or concentration of said at least one marker from one or more Nav channels is indicative of the efficacy of the neuroprotective treatment in the patient. Accordingly, the subject's therapeutic regime may be maintained or adjusted depending on the variation over the time of the level of the at least one marker as previously defined.

In one embodiment, the method for monitoring the treatment/rehabilitation efficacy of the patient includes the detection in a sample obtained from said patient of at least one marker from proteolytic cleavage of one or more sodium channel at 2 or more time points after spinal cord injury, wherein a lower level of said at least one marker at a later time point compared to a reference value obtained at an earlier time point (i.e., from a prior sample from said patient) is indicative of motor improvement; whereas a higher level of said at least one marker at a later time point compared to a reference value obtained at an earlier time point is indicative of motor outcome worsening.

In some conditions, an equal level of said at least one marker at a later time point compared to a reference value obtained at an earlier time point is indicative of motor stabilization.

In some embodiment, the present disclosure encompasses a method for treating a patient having SCI or suspected of having SCI, a method of selecting the most appropriate treatment to the patient with SCI or a method for adapting the treatment of a patient suffering from SCI, wherein said method comprises detection and measurement of the level of the at least one marker as previous defined (typically a-Nach BDP120) from said at least one Nav channel. An adapted neuroprotective treatment is thus further administered to patient diagnosed with SCI. In some embodiment the method comprises the determination of the clinical grade of SCI ( e.g ., patient staging) as previously detailed.

The method may further comprise the following steps:

- if the a-Nav-BDP120’ level of the at least one Nav is indicative of mild SCI, administering a treatment appropriate for mild SCI;

- if the level of the at least one Nav is indicative of severe SCI, administering a treatment appropriate for severe SCI.

It will be appreciated by those skilled in the art that different treatment pathways may be used. An appropriate treatment may include: neuroimaging techniques (particularly within 1 hour of injury); admission to a major trauma center with neurosurgical facilities; surgery; administering rehabilitation program, neuroprotective treatment and combinations thereof.

In some embodiments, the present disclosure also relates to a neuroprotective drug for use in a method of treatment of a patient suffering from spinal cord injury, wherein the methods comprises the determination of the clinical stage of spinal cord injury as previously described.

Neuroprotective treatments include, but are not limited to, methylprednisolone, 17a- estradiol, 17P-estradiol, ginsenoside, progesterone, simvastatin, deprenyl, minocycline, resveratrol, and other glutamate receptor antagonists (e.g. NMDA receptor antagonists) antioxidants, calpain-related therapeutic agents and combinations thereof.

Calpain-related therapeutic agents typically include, but are not limited to, ALLNal, MDL28170, AK295, Calpastatin, SNJ1945, BDA-410 ABT-957, Olesoxime,

Cyclosporine A, E-64d (EST, Estate, loxistatin and rexostatine), AK-295 (CX 295), C- 101 (myodur and CLA), CYLA, C201 (neurodur and CLA), CEP-3453, CEP-4143, Calpeptin (IPSI-001 ), A-705239 (BSF 409425), A-705253, Ala-1.0 and combination thereof. An appropriate treatment for mild SCI may include: removing the subject from activity; further evaluating the subject in hospital without overnight admission; or admission to hospital for a period of observation (typically 1 -2 days). The subject may be further evaluated using sensory and motor tests. CT scanning is generally only required if certain indications are present, including suspected spine fracture, focal neurological deficit, repeated vomiting.

An appropriate treatment for severe SCI may include: MRI or CT scanning (particularly within 1 hour of injury); admission to intensive care and/or transfer to a specialist clinic or major trauma centre with neurosurgical facilities; neuromonitoring; surgery; administering a therapy for alleviating SCI, such as administering neuroprotective drugs (listed above) and/or other neuroprotective measures, such as induced hypertension to enhance spinal cord perfusion.

Kits

The present invention also includes a kit for use in the methods as herein disclosed, wherein the kit comprises at least one reagent for detecting at least one marker as previously described (notably a-Nach BDP120) of one or more Nav channels and instructions for implementing said methods.

As used herein, a "reagent" refers to the components necessary for detecting or quantifying a marker as herein disclosed (and notably for detecting and measuring the level of a-Nach BDP120) from one or more Nav channels by any one of the methods described herein. For instance, kits can include reagents for performing liquid or gas chromatography, mass spectrometry, immunoassays, immunoblots, or electrophoresis to detect a-Nach BDP120 from one or more Nav channels as described herein.

The kit can comprise a labeled-binding partner that binds to a marker according to the present disclosure and notably to a-Nach BDP120 from one or more Nav channels, wherein said one or more Nav channels are selected from the group consisting of Nav1.1 , Nav1.2, Nav1.3, Nav1.4, Nav1.6, Nav1.7, Nav1.8 and Nav1.9 channels and combinations thereof. In some aspects, the binding partner can be one that does not specifically bind a-Nach BDP120 from one or more of Nav1.1 , Nav1.2, Navi .3, Navi .4, Navi .6, Navi .7, Navi .8 and Navi .9 channels (i.e., one that binds a subset or all a-Nav-BDP120’ from this group); or one that specifically binds a-Nav- BDP120’ from only one Nav of interest (a mono-specific antibody for instance). Suitable binding partners for specifically binding to Nav proteins include, but are not limited to, antibodies and fragments thereof, aptamers, peptides, and the like.

Labels that can be conjugated to the binding partner include metal nanoparticles (e.g., gold, silver, copper, platinum, cadmium, and composite nanoparticles), fluorescent labels (e.g., fluorescein, Texas-Red, green fluorescent protein, yellow fluorescent protein, cyan fluorescent protein, Alexa dye molecules, etc.), and enzyme labels (e.g., alkaline phosphatase, horseradish peroxidase, beta-galactosidase, beta- lactamase, galactose oxidase, lactoperoxidase, luciferase, myeloperoxidase, and amylase).

The kit may provide additional components that are useful in procedures, including, but not limited to, buffers, developing reagents, labels, reacting surfaces, means for detection, control samples, standards, instructions for implementing the method as per the present disclosure, and interpretive information.

This invention is further illustrated by the following additional examples that should not be construed as limiting. Those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made to the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.

EXAMPLES Materials and Methods Animals. Neonatal and adult (150-250 g) female Wistar rats (Charles River, Burlington MA USA) were used. Animals were housed in a temperature-controlled animal care facility with a 12 h light-dark cycle. Inventors made all efforts to minimize animal suffering and the number of animals used. Neonates were anesthetized by hypothermia. Inventors performed experiments in accordance with French regulations (Ministry of Food, Agriculture and Fisheries, Division of Health and Protection of Animals). The local Direction of Veterinary Services and Ethical Committee (Marseille, Provence) delivered the appropriate licenses and approved the protocols, respectively.

Surgery and postoperative care. Spinal cord transection or contusion were performed in adult rats. Adult female Wistar rats (220-280g) were anesthetized with ketamine (Imalgen, Merial, 50 mg/kg ip) and medetomidine (Domitor, Janssen, 0.25 mg/kg ip) and treated preventively with a long acting antibiotic (amoxicillin, Duphamox LA, Pfizer, 100 mg/kg s-c). A midline skin incision was performed and a local anesthetic (Proca^'ine chlorhydrate 2%, Pharmy H) was injected into the paravertebral muscles before retraction. A laminectomy of the T8 dorsal vertebra was performed carefully to expose the spinal cord. For the transection model, after cutting the dura, the spinal cord was completely transected with small scissors at the T9 segmental level under local anesthesia. For the contusion model, the rat was hang by T7 and T10, by using vertebral clamps and a 10 g rod was released from a height of 12.5 mm at the T8 level by using the NYU spinal cord injury device (New York University device). The rod trajectory was checked to ensure that all injury parameters are within the range. If any of the variables of impact (height: ± 0.5 mm; velocity ± 5 %), or the impact curve minimum (8 msec > impact), showed significant errors, the rat was excluded from the study. After transection or contusion of the spinal cord, the paravertebral muscles and skin were sutured and disinfected. Rats were rehydrated with 5ml of 0.9% NaCI s-c and kept warm in an incubator. Two hours later, they were awakened with atipamezol (Antisedan, Janssen, 0.12 mg/kg im) and injected with a morphinic analgesic (buprenorphine, Vetergesic, Sogeval, 0.05 mg. kg s-c, 3 injections at 8 h intervals to cover the first 24h). Rats were then placed in a warm room in clean individual cages with easily accessible complete natural food for rats (Vitakraft) and water bottles containing 1.33 g/l of aspirin for 3 days (Aspegic nourisson, Sanofi Aventis). Their bladder was emptied twice a day until recovery of autonomy. Their water intake, temperature and weight were checked every day until they recovered an ascending weight curve. After 7 days, they were housed again with their initial cage partners. Occasional urinary infection were treated with enrofloxacine (Baytril, Bayer, 5 mg/kg/j for 3 days).

In neonates, the procedure for the spinal cord transection was similar than that for adult animals except that animals were anesthetized by hypothermia and, the lesion cavity was filled with sterile absorbable local hemostat Surgicoll. The wound was then covered with Steri-Strips (3M Health Care, St. Paul, MN) and animals were kept warm and wet for 2 hours in cotton-wool swab impregnated with their mother smell before they returned to their home cage with their mother. The antibiotic amoxycilin (150 mg/kg, s.c.) was injected postoperatively. Sham animals were submitted to all procedures except the spinal cord transection.

Membrane protein isolation and Western blots. Tissues were collected from spinal cord lumbar enlargements and frozen after removing the dorsal and ventral roots. For the membrane fraction, corresponding to the plasma membrane-enriched fraction, samples were homogenized in ice-cold lysis buffer (320mM sucrose, 5mM Tris-HCL pH 7.5, 10mM iodoacetamide) supplemented with protease inhibitors (CompleteMini, Roche diagnostic Basel, Switzerland). Unsolubilized material was pelleted by centrifugation at 7,000g for 5 min. The supernatant was subjected to an additional centrifugation step at 18000g for 70 min at 4 °C. Pellets were collected and homogenized in ice cold lysis buffer (1 % Igepal CA-630, Phosphate Buffer Saline 1 X, 0.1 % SDS, 10mM iodoacetamide), supplemented with protease inhibitors (CompleteMini, Roche diagnostic). Protein concentrations were determined using a detergent-compatible protein assay (Bio-Rad, Hercules, CA, USA). Equal protein amounts (60pg) from samples were size fractionated by 6% (vol/vol) SDS/PAGE from 40% Acryl/Bisacrylamide (29/1 ) commercial solution, transferred to a PVDF membrane and probed with the sodium channel a-subunit lll-IV loop (pan:Sigma Aldrich, 1 :500) antibody or the all-spectrin (AA6 : Enzolife, 1 :1000) antibody at 4 °C overnight in Tris-buffered saline containing 5% fat-free milk powder. The blot was then incubated for 1 h at 22°C with an ImmunoPure goat HRP-conjugated mouse- specific antibody (1 :40,000 in blocking solution; Thermo Scientific, Waltham, MA, USA). The blots were blotted with an enhanced chemiluminescence detection (Merck-Millipore). Signal intensities were measured with the image analysis software Quantity-One (BioRad).

Plasma samples and western blots. Approximately 400mI of blood were taken from de tail vein of rats. Blood samples were homogenized in blood collection tubes (Microvette 500 EDTA-K3, Sarstedt) contening ice-cold PBS buffer supplemented with protease inhibitors (CompleteMini, Roche diagnostic Basel, Switzerland). Blood cells were pelleted by centrifugation at 3,000g for 10 min. The supernatant was collected and homogenized in ice-cold PBS buffer supplemented with protease inhibitors. Plasma can be stored at -80°c if it wasn't freshly using. Protein concentrations were determined using a detergent-compatible protein assay (Bio- Rad, Hercules, CA, USA). Equal protein amounts (80pg) from denatured samples were size fractionated by gradient 4-15% SDS/PAGE (mini protean TGX stain free gels, Biorad), transferred to a Nitrocellulose membrane and probed with the NaCh a- subunit lll-IV loop (pa Sigma Aldrich, 1 :500 [lot SLBL1663V]) antibody at 4 °C overnight in Tris-buffered saline containing 5% nonfat-dry milk powder (Blotting- Grade Blocker, Biorad). The blot was then incubated for 1 h at 22°C with an immunopure goat rat absorbed HRP-conjugated mouse-specific antibody (1 :5,000 in blocking solution; Clinisciences). The blots were blotted with an enhanced chemiluminescence detection (Merck-Millipore). Signal intensities were measured with the image analysis software Quantity-One (BioRad).

Results

Example 1. The level expression of a-Nav-BDP120’ positively correlates with the level expression of m-calpain in newborn and adult rats with SCI.

Pediatric patients are not spared from SCI. Suitable for this use is a biomarker expressed in the early developing spinal cord. In this context, experiments have been started in a newborn rat model. Calpains exist in the central nervous system as two major isoforms, m-calpain (or calpain 1 ) and m-calpain (or calpain 2) that differ from each other on the basis of the range of [Ca²⁺] that is required for their half-maximal activation (mM and mM ranges, respectively).

By means of immunoblotting, inventors measured the protein level of m- and m- calpain in the spinal cord within the first postnatal week after the spinal cord was transected at birth. The level expression of m-calpain probed by a polyclonal antibody was significantly increased in the spinal tissues after SCI compared to intact animals (Fig. 1 a,b). Similar results were obtained by using a m-calpain polyclonal antibody (Fig. 1 c,d). By contrast, the level expression of m-calpain did not change after SCI (Fig. 1 e,f). Inventors determined whether increase in expression of m-calpain was accompanied by an increase of the calpain proteolytic activity. Activation of calpain was assessed by quantifying the calpain-mediated cleavage of the Nav channels into a-Nav- BDP120’. Immunoblot analysis from control samples revealed low levels of a-Nav- BDP120’ (Fig 1 g,h). In contrast, immunoblot analysis of samples from rats with SCI showed an increase in the levels of a-Nav-BDP120’ when compared from control subjects (Fig. 1 g,h). Interestingly, the formation of a-Nav-BDP120’ is very much in parallel with the level expression of m-calpain (Fig. 1 i,j). Noteworthy a better correlation was obtained for the m-calpain monoclonal antibody (R=0.92) as compared to the m-calpain polyclonal antibody (R=0.52). Therefore, the monoclonal antibody was used for the following experiments. Similar positive relationship was also observed between the level expression of m-calpain and a-Nav-BDP120’ in adult rats with chronic SCI (R=0.96).

Altogether, these experiments show that protein levels of m-calpain in samples from both newborn and adult rats are increased following SCI and correlates with the proteolytic cleavage of Nav channels into a-Nav-BDP120’. In sum, a-Nav-BDPs120’ can serve as a relevant biomarker to assess the activation calpain following a SCI.

Example 2. Detection of a-Nav-BDP120’ expands the time frame to explore biomarkers for SCI. The timeline, or kinetics, of the emergence, persistence, and decline of the biomarkers is a rising area of active research. Acute biomarkers are valuable for confirming or ruling out a SCI shortly after a lesion. On the other hand, persistent biomarker level is essential to follow-up the progress of the lesion or to monitor the efficacy of a treatment.

Most biomarkers are rapidly elevated after injury. This is the case of BDPs from a-ll spectrin that have been reported in human to transiently increase in serum for up to 2 days after SCI but then decreases quite rapidly to baseline levels on the 3^rd day (Yokobori et al. , 2015). These results make the a-ll spectrin BDPs potential biomarkers for acute diagnosis of SCI but its utility values for patient with a chronic SCI remain low. It is important to find a novel biomarker of calpain activity both detectable shortly after SCI and traceable for months after SCI. The a-Nav-BDP120’ from Nav channels may present a new class of biomarkers for a chronic SCI as they remain quite a long time in situ, at least 4 months after injury (Brocard et al., 2016).

Here inventors investigated whether a-Nav-BDP120’ can also be used as a potential acute biomarker for SCI in adult rats. Samples were collected within 24 hours after a complete transection of the spinal cord, and then analyzed by immunoblot for levels of a-Nav-BDP120’ (Fig. 2a). The expression of a-Nav-BDP120’ significantly increased on the day of injury (Fig. 2a, b) and can be detectable as early as 3 hours post-SCI (Fig. 2c; the earliest time point of the investigation), while the level expression of full-length Nav channels (~250-kDa) decreased (Fig. 2a, b). This study shows that the cleavage of Nav channels is acutely elevated within the first hours following SCI showing a I role of a-Nav-BDP120’ in the early diagnosis of SCI.

Similar experiments have been performed in newborn rats. Inventors compare the time course expression BDPs from a-ll spectrin (Fig. 3a) to that of Nav channels (Fig. 3d) after a transection of the spinal cord at birth. In line with previous studies in adults, the time window for the detection of BDPs from a-ll spectrin was short after SCI. The BDPs from a-ll spectrin (~145kDa) rises within a few hours after SCI, but the level also came down quite fast to the normal level in 3-5 days post-SCI (Fig. 3c). At the same time, the a-Nav-BDP120’ were accumulated in sublesional spinal cord within the first day after injury, and the level remains quite steady for at least 6 days after injury (Fig. 3f). Note that neither the expression of full-length a-ll spectrin nor of Nav channels changes after SCI (Fig. 3b, e). Therefore, the kinetic profile of a-Nav- BDP120’ differs somewhat from that of many other biomarkers. While several biomarkers peak and then decline within a couple of days after injury, the concentration of a-Nav-BDP120’ still increases.

In sum, these results indicate that a- Nav-BDP120’ can serve as a short-term and a long-term biomarker of SCI both in adult and pediatric patients. Their fast occurrence after SCI can serve for rapid diagnostics while their long-lasting increase show a prognostic and monitor use in a clinical setting.

Example 3. Early detection of a-Nav-BDP120’ in adult rats with a mild SCI.

The need for quantitative and objective biomarkers of mild SCI has been emphasized by clinicians. Indeed, one difficulty in accurately diagnosing mild SCI relates to the frequent lack of radiological evidence to support the diagnosis, which often leads clinicians to diagnose mild SCI based on clinical symptoms known to overlap with other clinical conditions. For comparison purposes, rather than a complete transection of the spinal cord, a mild contusion was included in inventor’s investigation. Within the day of the mild SCI, a-Nav-BDP120’ increased (Fig. 4a, d) and remain elevated 48 hours post-SCI in adult rats (Fig. 4c, d). When data are pooled the increase was significant (Fig. 4d). The inventors also showed that a mild SCI releases a-Nav-BDP120’ in situ with a concomitant increase amount of p-calpain (Fig. 4e,f), thereby a clear linear relationship was observed between the level expression of m-calpain and the level expression of a-Nav-BDP120’ (Fig. 4g). The present invention is advantageous as the sensitivity of Nav channels to calpains after SCI appears to be high. Thus, the provided method can capture the vast majority of cases of SCI with a high predictive accuracy. In sum, detection of a-Nav-BDP120’ in samples from patients suspected of having a SCI can supplement diagnosis or perhaps serve as a new means of definitive acute diagnosis even for mild injuries.

Example 4. a-Nav-BDP from Navchannels may serve as potential biomarkers for SCI. The nine different Nav channel a subunit isoforms display heterogeneity in distribution, expression and function, yet all are greater than 50% identical in amino acid sequence in their transmembrane and extracellular domains. In particular, the pan-Nav antibody used in immunoblotting recognizes the conserved DIII-DIV linker of all Nav isoforms. In principle, all Nav isoforms are likely to be cleaved by calpain since all of them contain a calpain recruitment domain (PEST). Therefore, to define a protein signature associated with a SCI, it is important to see if some Nav channels are specifically cleaved after SCI. Inventors have focus their attention on Nav1.6 channel subtype predominantly expressed in spinal motoneurons. One week after a SCI, the immunoblotting of the spinal cord with the specific Navi .6-antibody revealed a prominent ~120-kDa band (Fig. 5a). These results show that calpain-mediated BDPs from Nav channels, such as Nav1.6 channels, can serve as potential biomarkers for SCI.

The inventors have further demonstrated that an increase of a-Nav channel fragments can be detected in the plasma of adult rats suffering from SCI both in acute (144 hours after the lesion, see figure 5b) and chronic stages (see figure 5 c,d). These results provide evidence that calpain-mediated BDPs from a subunit Nav channels (including for example the 120 kDa fragment obtained by calpain cleavage of the a-subunit in the ll-lll linker region), and in particular changes in the level of at least one fragment of at least one a Nav channel subunit, can be used as a biomarker based tools for diagnosis, prognosis and management of SCI and in particular a serum/plasma or whole blood biomarker

Example 5. a-Nav-BDP120’ to monitor the efficacy of promising neuroprotective therapies targeting calpains in the acute management of SCI and to predict motor outcome improvements.

It has long been recognized that much of the post-traumatic degeneration of the spinal cord following SCI is caused by a multi-factorial secondary injury process that occurs during the first hours and days after injury. In particular, the expression of calpains markedly increases in the acute phase following SCI (Banik et al., 1997; Springer et al., 1997; Schumacher et al., 1999). Also, a single intraspinal microinjection of the potent calpain blocker MDL28170 after acute SCI significantly improves motor outcomes (Yu et al., 2008). These experimental evidence support the possibility that treatments targeting calpains in animal with acute SCI might be neuroprotective. Investigations from examples 3 and 4 show the early expression of a-Nav-BDP120’ after SCI. Here, inventors investigated if a short-lasting administration of MDL28170 in rats with acute SCI is accompanied by a decrease in the level expression of a-Nav-BDP120\ After the SCI, adult rats received a daily i.p. injection of MDL28170 (10 mg/kg for two days beginning within the hour after SCI). Such treatment prevented the significant increase of Nav channels proteolysis SCI as confirmed by a lower amount of a-Nav-BDP120’ in MDL28170-treated rats (Fig. 6a, b). Thus, a brief pharmacological inhibition of calpain by MDL28170 soon after the SCI reduces the cleavage of Nav channels. In sum, a-Nav-BDP120’ can monitor the efficacy of promising neuroprotective therapies targeting calpains in the acute management of SCI. Because such treatment alleviates upcoming motor deficits following SCI, changes in the level expression a-Nav-BDP120’ can also predict motor outcomes. In this example, a decrease in the level expression of a-Nav-BDP120’ suggests that the patient is a good responder to the treatment and that the patient's health is likely to improve.

Example 6. a-Nav-BDP120’ to monitor the efficacy of promising therapies targeting calpains in the chronic management of SCI and to predict motor outcome improvements.

Tetracyclines have been used safely as an antibiotic agent for many years in the clinic. The properties of clinical tolerance and easy penetration into the brain make some of the tetracycline derivatives potential therapeutic reagents for neuroprotection (Klein and Cunha, 2001 ). Among them, demeclocycline (DMC) is neuroprotective against glutamate toxicity in cultured mouse of cerebellar neurons and focal cerebral ischemia in vivo (Jiang et al., 2005). Interestingly, the effect involves a direct and a potent inhibition of m- and m-calpains. Notably, the patent W02005082860A1 claims the use of tetracyclines in treating conditions implicated by or associated with calpain activity or activation. Altogether, this suggests that treatment with DMC in the chronic phase may have positive effects for reducing calpain activity after SCI. Rats with chronic SCI were divided into those that received DMC treatment (45 mg/kg every 12 h for 5 days) and those who did not. As shown in Figure 6, the data indicate that the levels of a-Nav-BDP120’ were lower in the DMC-treated group when compared to the untreated group (Fig. 6, c,d). Thus post-traumatic therapeutic with DMC lowers the levels of calpain activation in adult rats with chronic SCI, consistent with preclinical and clinical findings that patients that receive the anti-inflammatory treatments with tetracyclines present better motor outcomes when compared to those patients who do not get the treatment (Ulndreaj et al., 2017). In sum, a-Nav-BDP120’ is efficient to monitor the efficacy of promising therapies targeting calpains in the chronic management of SCI and to predict motor benefits.

Example 7. a-Nav-BDP120’ to monitor the efficacy of promising therapies alleviating spasticity in pediatric patients with SCI.

In the healthy spinal cord, the excitability of motoneurons is set by brainstem-derived serotonin (5-HT) but a direct implication of brainstem serotonergic neurons in the development of spasticity, has not been explored so far. Over the last decade, inventors’ studies have focused on the contribution of 5-HT projections to the maturation of spinal motoneurons and networks. The major conclusion of these studies and the leading thread of forthcoming experiments suggests that abnormalities in motor control are rooted in 5-HT depletion. Specifically, it appears from animals depleted in 5-HT, that a marked increase in the excitability of lumbar networks occurs (Pflieger et al., 2002), similar to that observed when spasticity emerges after SCI (Norreel et al., 2003). Consistent with this, the degeneration of brainstem 5-HT neurons triggers spasticity in amyotrophic lateral sclerosis (El et al., 2017). The wide range of drugs targeting the 5-HT system could be useful to treat spasticity in neurological diseases. Notably, inventors have demonstrated that the specific activation of 5-HT_2a receptors after SCI reduces spasticity (Bos et al., 2013). Here inventors investigated whether a-Nav-BDP120’ channels can also be used to monitor treatment efficacy in alleviating spasticity in pediatric patients with SCI. When newborn rats with SCI are treated with DOI to specifically activate 5-HT₂ receptors, the level of a-Nav-BDP120’ was lowered thereby the significant increase of a-Nav-BDP120’ after SCI was prevented (Fig. 6 e,f). This leads inventors to consider the hypothesis that the lack of 5-HT in the sublesional spinal cord may be one of the primary mechanisms by which spasticity develops after SCI, likely through a calpain-mediated mechanism. These results alsoshow that a-Nav-BDP120’ is efficient to monitor the efficacy of promising therapies alleviating spasticity in the management of pediatric patients with SCI and to predict motor benefits.

Altogether, examples 5, 6 and 7 show that levels of a-Nav-BDP120’ can serve as objective, biochemical indicators of treatment efficacy for patients with SCI and can predict an unfavorable versus favorable outcome. Such markers can also direct treatment and rehabilitation efforts. The clinician would target therapies to patients identified as having a greater risk of inflammation-mediated secondary injury.

REFERENCES

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Claims

Claims

1. The use of one or more proteolytic fragments of one or more sodium channels as a biomarker

- for diagnosing spinal cord injury in a patient,

- for assessing the clinical grade of spinal cord injury or predicting the motor outcome of a patient suffering from spinal cord injury,

- for predicting the efficacy of a treatment for spinal cord injury in a patient, or

- for monitoring the motor outcome or treatment efficacy in a patient suffering from spinal cord injury; wherein said one or more proteolytic fragments are detected in a serum, whole blood or plasma sample from said patient.

2. The use according to any one of the preceding claims, wherein the sodium channel is selected from Nav1.1 , Nav1.2, Nav1.3, Nav1.6, Nav1.7, Nav1.8 and Navi .9.

3. The use according to any one of the preceding claims, wherein the spinal cord injury result from traumatic injury, ischemic neuropathies or compression syndrome.

4. The use according to any one of the preceding claims, wherein the level of sodium channel cleavage fragments is measured by liquid chromatography, gas chromatography, mass spectrometry, radioimmunoassays, immunofluorescent assays, FRET-based assays, immunoblot, ELISAs, or liquid chromatography followed by mass spectrometry.

5. The use according to any one of the preceding claims, wherein the patient is a mammalian, notably a human and typically a pediatric patient.

6. The use according to any one of the preceding claims, for assessing the clinical grade of spinal cord injury or predicting motor outcome in a patient suffering from spinal cord injury or suspected to suffer from spinal cord injury comprising the detection in a sample obtained from said patient of at least one marker from proteolytic cleavage of one or more sodium channels, wherein the level of said at least one marker is indicative of the clinical grade or the motor outcome of the patient.

7. The use according to any one of claims 1 -5, for diagnosing spinal cord injury, comprising the detection in a sample obtained from said patient of at least one marker from proteolytic cleavage of one or more sodium channels, wherein an increase of said marker as compared to a control, is indicative of spinal cord injury.

8. The use according to any one of claims 1 -5 for predicting the efficacy of a treatment for spinal cord injury in a patient comprising the detection in a sample obtained from said patient of at least one marker from proteolytic cleavage of one or more sodium channels, wherein a decrease of said marker as compared to a control, is indicative of treatment efficacy.

9. The use according to any one of claims 1 -5, for monitoring the motor outcome or treatment efficacy in a patient suffering from spinal cord injury comprising the detection in a sample obtained from said patient of at least one marker from proteolytic cleavage of one or more sodium channel at 2 or more time points after spinal cord injury, wherein a decrease of said at least one marker as compared to a control is indicative of motor improvement or of treatment efficacy.

10. A method for assessing the clinical grade of spinal cord injury or predicting motor outcome in a patient suffering from spinal cord injury or suspected to suffer from spinal cord injury comprising the detection in a sample obtained from said patient of at least one marker from proteolytic cleavage of one or more sodium channel, wherein the level of said at least one marker is indicative of the clinical grade or the motor outcome of the patient.

1 1. A method for diagnosing spinal cord injury in a patient comprising the detection in a sample obtained from said patient of at least one marker from proteolytic cleavage of one or more sodium channel, wherein an increase of said at least one marker as compared to a control is indicative of spinal cord injury.

12. A method for predicting the efficacy of a treatment for spinal cord injury in a patient comprising the detection in a sample obtained from said patient of at least one marker from proteolytic cleavage of one or more sodium channel, wherein a decrease of said at least one marker as compared to a control is indicative of treatment efficacy.

13. A method for monitoring the motor outcome or treatment efficacy in a patient suffering from spinal cord injury comprising the detection in a sample obtained from said patient of at least one marker from proteolytic cleavage of one or more sodium channel at 2 or more time points after spinal cord injury, wherein a decrease of said at least one marker as compared to a control is indicative of motor improvement.

14. A neutroprotective drug for use in a method of treatment of a patient suffering from spinal cord injury, wherein the method comprises the determination of the clinical stage of spinal cord injury according to claim 10.

15. The neutroprotective drug for use according to claim 14, wherein said neuroprotective drug is selected from methylprednisolone, 17a-estradiol, 17P- estradiol, ginsenoside, progesterone, simvastatin, deprenyl, minocycline, resveratrol, other glutamate receptor antagonists, antioxidants, calpain-related therapeutics agents and combination thereof.

16. The neutroprotective drug for use according to claim 15, wherein calpain- related therapeutic agents include ALLNal, MDL28170, AK295, Calpastatin, SNJ1945, BDA-410 ABT-957, Olesoxime, Cyclosporine A, E-64d (EST, Estate, loxistatin and rexostatine), AK-295 (CX 295), C-101 (myodur and CLA), CYLA, C201 (neurodur and CLA), CEP-3453, CEP-4143, Calpeptin (IPSI-001 ), A-705239 (BSF 409425), A-705253, Ala-1.0, and combination thereof.

17. A kit for use in a use according to any one of claims 1 -9 or in method according to any one of claims 10-13, wherein the kit comprises a reagent for detecting sodium channel cleavage fragments and instructions for implementing said methods.