METHODS AND COMPOSITIONS FOR THE TREATMENT OF INTERVERTEBRAL DISC HERNIATION

Abstract

The invention relates to methods and compositions for use in the treatment or prophylaxis of intervertebral disc herniation in a mammal. The composition according to the invention comprises an inhibitor of T-cell activation, said inhibitor being capable of inhibiting CD28-mediated co-stimulation of T-cells. The said inhibitor of T-cell activation is preferably a protein comprising either an exact or a modified version of the extracellular domain of CTLA-4, such as abatacept, belatacept, XPro9523 and/or ASP2408.

Claims

1-18. (canceled)

19. A method for the treatment or prophylaxis of intervertebral disc herniation, comprising administering to a mammal in need thereof a therapeutically effective dose of an inhibitor of CD28-mediated co-stimulation of T-cells, which inhibitor is a protein comprising at least one extracellular domain of CTLA-4; wherein the treatment or prophylaxis comprises (a) reduction or prevention of disc hernia formation; (b) reduction of disc hernia size, or both.

20. The method according to claim 19 wherein the inhibitor of CD28-mediated co-stimulation of T-cells is a protein which is capable of binding to CD80/CD86.

21. (canceled)

22. The method according to claim 19 wherein the protein is a fusion protein comprising the Fc region of immunoglobulin G (IgG) fused to the at least one extracellular domain of CTLA-4.

23. The method according to claim 19 wherein the extracellular domain of CTLA-4 is chosen from: (a) a polypeptide having an amino acid sequence comprising the sequence shown as SEQ ID NO: 3 or SEQ ID NO: 4; (b) a polypeptide having an amino acid sequence which is at least 90% identical with the sequence shown as SEQ ID NO: 3 or SEQ ID NO: 4; (c) a polypeptide having an amino acid sequence consisting essentially of the sequence shown as SEQ ID NO: 3 or SEQ ID NO: 4; or (d) a polypeptide having an amino acid sequence consisting of SEQ ID NO: 3 or SEQ ID NO: 4.

24. The method according to claim 22 wherein the fusion protein is a dimer comprising two monomers chosen from: (a) a polypeptide having an amino acid sequence comprising the sequence shown as SEQ ID NO: 1 or SEQ ID NO: 2; (b) a polypeptide having an amino acid sequence which is at least 90% identical with the sequence shown as SEQ ID NO: 1 or SEQ ID NO: 2; (c) a polypeptide having an amino acid sequence consisting essentially of the sequence shown as SEQ ID NO: 1 or SEQ ID NO: 2; or (d) a polypeptide having an amino acid sequence consisting of SEQ ID NO: 1 or SEQ ID NO: 2.

25-27. (canceled)

28. The method according to claim 19, wherein the treatment or prophylaxis of intervertebral disc herniation comprises reduction of inflammation.

29-30. (canceled)

31. The method according to claim 19, wherein the treatment or prophylaxis of intervertebral disc herniation comprises reduction or prevention of the formation of granulation tissue.

32. The method according to claim 19, wherein the intervertebral disc herniation comprises extrusions, sequestrations, or both.

33. The method according to claim 19, wherein the composition is administered intravenously or subcutaneously.

34. The method according to claim 19, wherein the inhibitor of CD28-mediated co-stimulation of T-cells is administered in a dose of between 0.1-20 mg/kg body weight.

35. The method according to claim 19, wherein the inhibitor of CD28-mediated co-stimulation of T-cells is administered by subcutaneous injection once weekly in a dose of 125 mg.

36. The method according to claim 19, wherein the mammal is a human.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0021] FIG. 1: Nodule size as determined by objective measurements of MRI images for the abatacept study. The chart displays mean values with error bars representing+1 SD. Asterisks mark differences of statistical significance (*=p0.05, **=p0.01).

[0022] FIG. 2: Nodule size as determined by subjective estimation of MRI images for the abatacept study. The chart displays mean values with error bars representing+1 SD. Asterisks mark differences of statistical significance (*=p0.05, **=p0.01).

[0023] FIG. 3: Nodule size as determined by macroscopic analysis after euthanization and dissection upon termination of the study (week 8), abatacept study. 0=no nodule, 1=small nodule, 2=clear nodule, 3=pronounced nodule. The chart displays mean values with error bars representing+1 SD. Significant differences are indicated with asterisks (*=p=0.005, **=p=0.001).

[0024] FIG. 4: Spontaneous resorption mechanism cascade as proposed by Kato et al. An inflammatory reaction starts when macrophages are activated upon contact with disc tissue, causing an upregulated TNF expression. TNF induces Vascular Endothelial Growth Factor (VEGF) and MMP's. TNF also induces plasminogen activators (PA) generating plasmin which in turs also activates MMP's. MMP's are then responsible for the degradation of the herniated disc matrix leading to spontaneous resorption.

DESCRIPTION OF THE INVENTION

[0025] It has surprisingly been found that inhibitors of CD28-mediated co-stimulation of T-cells are useful in the treatment and prophylaxis of intervertebral disc herniation in mammals. According to the invention, inhibition of CD28-mediated co-stimulation of T-cells prevents formation of disc hernias; as well as causes the disc hernia to decrease in size, thus relieving the nerve root from pressure. This is in sharp contrast to the previous belief that autoimmunity contributes to spontaneous resorption. Further, inhibition of CD28-mediated co-stimulation of T-cells is likely to reduce inflammation and thus cytokines known to be important for the sensitization of the nerve root.

[0026] Consequently, in a first aspect the invention provides a composition for use in the treatment or prophylaxis of intervertebral disc herniation in a mammal, such as a human, said composition comprising a modulator of T-cell co-stimulation, such as an inhibitor of T-cell activation. The term treatment or prophylaxis as used herein includes (i) preventing the disease, i.e. causing the clinical symptoms of the disease not to develop; (ii) inhibiting the disease, i.e. arresting the development of clinical symptoms; and/or (iii) relieving the disease, i.e. causing the regression, amelioration or elimination of clinical symptoms.

[0027] Preferably, the said inhibitor of T-cell activation is a protein, which is capable of binding to CD80/CD86. The term CD80/CD86 refers to the proteins Cluster of Differentiation 80 (also known as B7-1) and Cluster of Differentiation 86 (also known as B7-2), which work in tandem to prime T-cells. The said proteins are found on antigen-presenting cells (APCs) and provide costimulatory signals necessary for T-cell activation and survival. CD80/CD86 are ligands for two different proteins on the T-cell surface: CD28 (for autoregulation and intercellular association) and CTLA-4 (for attenuation of regulation and cellular disassociation).

[0028] More preferably, the said inhibitor of T-cell activation is a protein comprising at least one extracellular domain of CTLA-4 (cytotoxic T-lymphocyte-associated protein 4; also known as CD152 (cluster of differentiation 152)). Specifically, the said protein could comprise one extracellular domain or, preferably, two extracellular domains of CTLA-4. CTLA-4 is a protein receptor, which transmits an inhibitory signal to T cells when bound to CD80 or CD86 on the surface of antigen-presenting cells.

[0029] The said protein comprising the extracellular domain of CTLA-4 is preferably a fusion protein comprising the fragment crystallizable (Fc) region of IgG fused to the extracellular domain of CTLA-4. The said protein could be a monomer or, preferably, a dimer of such a fusion protein. The said immunoglobulin G can be selected from any subclass of IgG (IgG1, IgG2, IgG3 and IgG4). Preferably, the said IgG is IgG1. Alternatively, the fusion protein can comprise the Fc region of other immunoglobulins, including IgA1, IgA2, IgD, IgE and IgM.

[0030] The said extracellular domain of CTLA-4 is preferably chosen from: [0031] (a) a polypeptide having an amino acid sequence comprising the sequence shown as SEQ ID NO: 3 or SEQ ID NO: 4; [0032] (b) a polypeptide having an amino acid sequence which is at least 90% identical, preferably at least 95%, 96%, 97%, 98% or 99% identical, with the sequence shown as SEQ ID NO: 3 or SEQ ID NO: 4; [0033] (c) a polypeptide having an amino acid sequence consisting essentially of the sequence shown as SEQ ID NO: 3 or SEQ ID NO: 4; or [0034] (d) a polypeptide having an amino acid sequence consisting of the sequence shown as SEQ ID NO: 3 or SEQ ID NO: 4.

[0035] Preferably, the said fusion protein is a dimer comprising two identical or non-identical monomers. Each monomer in the said fusion protein is preferably chosen from: [0036] (a) a polypeptide having an amino acid sequence comprising the sequence shown as SEQ ID NO: 1 or SEQ ID NO: 2; [0037] (b) a polypeptide having an amino acid sequence which is at least 90% identical, preferably at least 95%, 96%, 97%, 98% or 99% identical, with the sequence shown as SEQ ID NO: 1 or SEQ ID NO: 2; [0038] (c) a polypeptide having an amino acid sequence consisting essentially of the sequence shown as SEQ ID NO: 1 or SEQ ID NO: 2; or [0039] (d) a polypeptide having an amino acid sequence consisting of the sequence shown as SEQ ID NO: 1 or SEQ ID NO: 2.

[0040] The polypeptides shown as SEQ ID NO: 1 and 2 represent monomers of the fusion proteins known as abatacept and belatacept, respectively. The polypeptides shown as SEQ ID NO: 3 and 4 represent the extracellular part of CTLA4 used in abatacept and belatacept, respectively.

[0041] Alternatively, the said inhibitor of CD28-mediated co-stimulation of T-cells is a protein comprising affinity and antagonistic properties towards CD28. More specifically, the said protein could comprise a monovalent pegylated Fab Ab antagonist of CD28, such as FR104.

[0042] Other known modulators and/or inhibitors of CD28-mediated co-stimulation of T-cells include AB103, anti-CD86 Monoclonal Antibody AIDA, BMS931699, CTLA4Ig MEDEXGEN, Debio0615; RG2077, and ShK186. Further, the said inhibitor of CD28-mediated co-stimulation of T-cells could be a protein comprising affinity and antagonistic properties towards CD86, such as ES210 or PG140, or towards CD80, such as IDEC114, KNO18, KNO19, Maxy30, or RhuDex.

[0043] In the present context, the term comprising should not be understood to be exclusively restricted to the recited amino acid sequences, instead it should be understood by the open-ended meaning including or containing. Consequently, the term comprising does not exclude sequences having additional, unrecited amino acids.

[0044] The identity of a sequence with a reference sequence refers to the percent of residues that are the same when the two sequences are aligned for maximum correspondence between residues positions. More specifically, the sequence identity expressed in percentage is defined as the value determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the sequence in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity. Unless indicated otherwise, the comparison window is the entire length of the sequence being referred to. In this context, optimal alignment is the alignment produced by the BLASTP algorithm as implemented online by the US National Center for Biotechnology Information (see The NCBI Handbook [Internet], Chapter 16), with the following input parameters: Word length=3, Matrix=BLOSUM62, Gap cost=11, Gap extension cost=1.

[0045] The term consisting essentially of is intended to exclude that which materially changes basic and novel characteristics of the invention. Consequently, polypeptides having an amino acid sequence consisting essentially of the sequence shown as SEQ ID NOS: 1 to 4 encompasses polypeptides carrying modifications like substitutions, small deletions, insertions or inversions, which polypeptides nevertheless have substantially the biological activities of the polypeptides shown as SEQ ID NOS: 1 to 4.

[0046] It is envisaged that other components of the adaptive immune system could be used according to the invention, either on their own, or in combination with each other or with T-cell inhibitors as defined above. Such components include, but are not limited to, various interleukin inhibitors such as anakinra, tocilizumab, basiliximab, ustekinumab, kanakinumab, sekukinumab, siltuximab; TNF-inhibitors such as etanercept, infliximab, afelimomab, adalimumab, golimumab, cetrolizumabpegol; calcineurin inhibitors such as ciclosporin or tacrolimus; B-cell inhibitors such as rituximab; or other immunomodulating drugs with effects on the adaptive immune system such as immunoglobulin therapy, glucocorticoids, methotrexate, leflunomide, sulfasalazine, azathioprine, cyclophosphamide or doxycycline.

[0047] As mentioned above, the composition for use according to the invention is useful in the treatment or prophylaxis of intervertebral disc herniation. The terms disc herniation and disc hernia are to be understood as known in the art, see e.g. Fardon et al. [50]. Consequently, herniation is broadly defined as a localized or focal displacement of disc material beyond the limits of the intervertebral disc space. The presence of disc tissue extending beyond the edges of the ring apophyses, throughout the circumference of the disc, is called bulging. Herniated discs may be classified as protrusion or extrusion, based on the shape of the displaced material. Protrusion is present if the greatest distance between the edges of the disc material presenting outside the disc space is less than the distance between the edges of the base of that disc material extending outside the disc space. The base is defined as the width of disc material at the outer margin of the disc space of origin, where disc material displaced beyond the disc space is continuous with the disc material within the disc space. Extrusion is present when, in at least one plane, any one distance between the edges of the disc material beyond the disc space is greater than the distance between the edges of the base of the disc material beyond the disc space or when no continuity exists between the disc material beyond the disc space and that within the disc space. The latter form of extrusion is best further specified or subclassified as sequestration if the displaced disc material has lost continuity completely with the parent disc. For further information, see reference [50] and drawings therein.

[0048] Consequently, the term treatment or prophylaxis of intervertebral disc herniation should be understood as comprising at least one, and preferably two, three, four or five, of the following effects: [0049] (i) reduction or prophylaxis of inflammation; [0050] (ii) reduction or prophylaxis of the formation of disc herniations, including protrusions, extrusions and sequestrations; and [0051] (iii) reduction of the size of disc herniations, including protrusions, extrusions and sequestrations; [0052] (iv) reduction or prophylaxis of bulge formation; and [0053] (v) reduction of the size of disc bulges.

[0054] In this context the term reduction is intended to encompass partial reduction, as well as complete inhibition or elimination of the said conditions.

[0055] As mentioned above in the Background Art section, human disc hernias have been found to comprise various types of tissue, including nucleus pulposus, annulus fibrosus, cartilaginous endplate and granulation tissue (scar tissue). The relative amount of granulation tissue has been found to be higher in younger patients. Granulation tissue has also been found to be more present in sequestrations and extrusions as compared to bulges and protrusions. The compositions for use according to the invention are useful in the treatment of disc hernias of all the said forms, and may be particularly useful for the treatment of younger patients with disc extrusions or sequestrations. Consequently, in a preferred aspect the invention provides compositions, as disclosed herein, for use in the treatment or prophylaxis of intervertebral disc herniation in a mammal, wherein the said treatment or prophylaxis comprises reduction or prevention of the formation of granulation tissue, especially wherein the said disc herniation comprises extrusions and/or sequestrations.

[0056] Further, the term intervertebral disc herniation includes conditions characterized by radiating pain and/or back pain, which conditions are deemed by a physician likely to be caused by intervertebral disc herniation, with or without radiological confirmation with e.g. MRI. Moreover, the intervertebral disc herniation can be located at various locations in the spine, all of which are included in this definition, including cervical, thoracic and lumbar intervertebral disc herniation.

[0057] The composition for use according to the invention can be administered to the patient in various ways, such as intravenously, intramuscularly, subcutaneously, intraperitoneally, orally, topically, rectally, etc. Further, the composition can be administered by a slow-release device, such as a miniosmotic pump implanted into the subject's body.

[0058] For instance, when the composition comprises a fusion protein such as abatacept, belatacept, ASP2408 or XPro9523, including combinations thereof, it could be administered in accordance with the methods known in the art for the said compounds. For example, the fusion protein could be administered as intravenous infusion in a dose of 0.1-20 mg/kg body weight, such as 0.5-20, 1-20, 5-20, or 5-15 mg/kg body weight, or more preferably around 10 mg/kg body weight. Alternatively, fusion protein could be administered in a dose of 500 mg for patients below 60 kg body weight; 750 mg for patients 60 to 100 kg body weight; or 1000 mg for patients above 100 kg body weight. Following the initial intravenous administration, an intravenous infusion could be given at 2 and 4 weeks after the first infusion and every 4 weeks thereafter.

[0059] Alternatively, the composition could be administered by subcutaneous injection, e.g. in a dose of 125 mg fusion protein once weekly. Such treatment may be initiated with or without an intravenous loading dose. For patients initiating therapy with an intravenous loading dose, treatment could be initiated with a single intravenous infusion, followed by the first 125 mg subcutaneous injection administered within a day of the intravenous infusion.

[0060] Further, the composition could be formulated such that it may be administered in a therapeutically effective amount by local injection to an intervertebral disc.

[0061] In a further aspect, the invention provides a method for the treatment or prophylaxis of intervertebral disc herniation, comprising administering to a mammal, such as a human, in need thereof a therapeutically effective dose of an inhibitor of T-cell activation. Effects and features of this second aspect of the present invention are analogous to those described above in relation to the first aspect of the present invention. In particular, the said inhibitor of T-cell activation is preferably a fusion protein, such as abatacept or belatacept, comprising the Fc region of immunoglobulin IgG1, fused to the extracellular domain of CTLA-4.

EXAMPLES

Example 1: Pilot Study, Treatment with Abatacept

[0062] 20 female Sprague-Dawley rats weighing approximately 225 g were evenly divided into two groups: disc puncture (DP, n=10) and disc puncture+abatacept (DP+abatacept, n=10). The rats were housed with free access to food and water in environmental-enriched cages. All rats underwent the same surgical procedure as described below.

[0063] Anesthesia was induced and maintained by the inhalation of Isoflurane. A single dose of 0.05 mg/kg buprenorphine (Temgesic) was administered subcutaneously before the procedure for per- and postoperative analgesia.

[0064] Following induction of anesthesia, a skin incision of approximately 6 cm was made in the midline over the spinous processes of the lumbar and caudal spine. The spinal muscles on the left side of the lumbar spine were dissected to expose the left L4/L5 facet joint. The left L4/L5 facet joint was removed to expose the underlying dural sac, L4 nerve root and L4/L5 intervertebral disc. The disc was then punctured using a 23 g needle. A small amount of air (0.2-0.3 ml) was injected into the disc to facilitate leakage of nucleus pulposus. The spinal muscles and the thoracolumbar fascia were then sutured and the skin closed with metal clips.

[0065] For the DP group, no additional treatment was given. For the DP+abatacept group, 10 mg/kg abatacept was administered intraperitoneally immediately before surgery. This was repeated 7 and 14 days after surgery.

[0066] To evaluate the effects on the morphology of the hernia-like nodule a macroscopic analysis was performed. 21 days after surgery anesthesia was again induced by inhalation of isoflurane followed by euthanization by incision of the heart, causing the rat to quickly bleed to death. The lumbar spine was then dissected by removing the spinal muscles and the vertebral arches of L4 and L5, exposing the L4/L5 intervertebral disc. A macroscopic analysis was performed through the surgical microscope by an experienced researcher. A description of the different types of data collected can be found in Table 1.

[0067] The data from the macroscopic analysis was considered ordinal. To test for statistical significance between the DP and DP+abatacept groups, Mann-Whitney U-tests were performed. Significance was defined as p0.05.

[0068] All rats survived the surgery and displayed good general condition throughout the study. In the DP group, 8 out of 10 rats displayed clear or pronounced disc nodules (Table 2). In the DP+abatacept group, no clear or pronounced nodules were found. The difference in nodule size was significant between the two groups (p=0.000). There was also significantly less inflammation observed in the DP+abatacept group (p=0.005). Tendencies of less osteophytes were also noted, but this difference was not significant (p=0.143).

Example 2: Longitudinal Assessment of Nodule Size, Treatment with Abatacept

[0069] The purpose of this study was to assess 1) how the nodule formed after disc puncture in the rat varies in size over time, 2) if the formation of the nodule can be inhibited by administration of the T-cell inhibitor abatacept, and 3) if the size of existing nodules can be reduced by administration of the T-cell inhibitor abatacept.

[0070] 24 female Sprague-Dawley rats (n=24) weighing approximately 225 g were evenly divided into three groups: Control group (no treatment, n=8); Treatment start day 0 (n=8); and Treatment start day 14 (n=8). The rats were housed with free access to food and water in environmental-enriched cages. All rats underwent the same surgical procedure as described below.

[0071] Anesthesia was induced and maintained by the inhalation of Isoflurane. A single dose of 0.05 mg/kg buprenorphine (Temgesic) was administered subcutaneously before the procedure for per- and postoperative analgesia.

[0072] Following induction of anesthesia, a skin incision of approximately 6 cm was made in the midline over the spinous processes of the lumbar and caudal spine. The spinal muscles on the left side of the lumbar spine were dissected to expose the left L5/L6 facet joint. The left L5/L6 facet joint was removed to expose the underlying dural sac, L5 nerve root and L5/L6 intervertebral disc. The disc was punctured using a 23 g needle. A small amount of air (0.2-0.3 ml) was injected into the disc to facilitate leakage of nucleus pulposus. The spinal muscles and the thoracolumbar fascia were then sutured and the skin closed with metal clips. The researcher performing the surgery was blinded to treatment groups.

[0073] All rats received an intraperitoneal injection immediately before surgery and subsequently once a week throughout the study period. For the different groups, the compound administered at the different time periods were as shown in Table 3.

[0074] For dosage calculations, a body weight of 225 g was assumed for all rats throughout the study period. When abatacept was not administered, saline was administered for blinding purposes. The rats were shortly anesthetized by inhalation of isoflurane before each injection to allow for a safe administration of the compound and to minimize pain and stress to the animal.

[0075] To assess the size of the disc nodules following disc puncture, MRI images were obtained with a 7T MRI system (Bruker Bio Spec) week 1, 2, 4 and 8 after surgery. The rats were anesthetized by inhalation of isoflurane throughout the MRI measurements. During the measurements, breathing frequency was monitored and the body temperature maintained by placing the rat on a heated pad.

[0076] A sagittal T2-weighted 3D RARE sequence with isotropic voxels of 150 m was used. The volume of the nodule was measured by manually defining a region of interest (ROI) of the disc aggrandizement dorsal to a longitudinal line between the most dorsal aspects of the adjacent vertebrae on each image where the disc could be identified. The number of voxels within the ROI was assessed three times for each image. The average number of voxels for each sagittal section of the disc were added and then multiplied by the voxel volume (0.003375 mm.sup.3), resulting in the total nodule size expressed in mm.sup.3. In addition to this, a subjective assessment of the nodule size was also performed by the observer for each MM. The researcher collecting the data was blinded to treatment groups.

[0077] Upon termination of the study, a macroscopic analysis was performed in addition to the MRI measurements. After the final MRI measurement 8 weeks after surgery the rats were euthanized by incision of the heart while still anesthetized by inhalation of isoflurane, causing the rat to quickly bleed to death. The lumbar spine was dissected by removing the spinal muscles and the vertebral arches of L5 and L6, exposing the L5/L6 intervertebral disc. A macroscopic assessment of the nodule size was performed through the surgical microscope. The assessments of the nodule size were performed according to the description in Table 1. The observer was not blinded to treatment groups for logistic reasons.

[0078] All rats survived the surgery and displayed good general condition throughout the study. The data from the macroscopic analysis and the subjective assessment from the MRI were considered ordinal. The data from the volume measurement on the MM was considered continuous Mann-Whitney U-tests were performed, and statistical significance defined as p0.05.

[0079] The results from the MM measurements can be found in FIG. 1 and FIG. 2. The results from the macroscopic analysis can be found in FIG. 3. The nodule was found to be significantly smaller in both treatment groups as compared to control by all types of assessments on week 4 and 8. In addition, a significant difference in nodule size as determined by subjective assessment was noted between Control (no treatment) and Treatment start day 0 on week 2.

[0080] To conclude, the results of this study confirmed that administration of abatacept inhibited the formation of the hernia-like nodule when treatment started immediately before disc puncture. Also, when treatment with abatacept was initiated two weeks after disc puncture, the hernia-like nodules that had formed during the two weeks became significantly smaller compared to the control group, indicating that abatacept can also cause shrinkage of the hernia-like nodule after it has formed. These results contradict the generally accepted hypothesis that autoimmunity and inflammation are beneficial for the resorption of hernias, but rather suggest that specific inhibition of the immune system, more specifically T-cells, can cause resorption and thus shrinkage of hernias.

Example 3: Longitudinal Assessment of Nodule Size and Immunohistochemistry, Treatment with Specific Inhibitor of CD28-Mediated Co-Stimulation of T-Cells

[0081] The purpose of this study is 1) to reproduce previous results indicating that treatment with a specific inhibitor of CD28-mediated co-stimulation of T-cells can be used to induce and/or expedite resorption of disc hernias in the modified animal model described in Example 3, and 2) to make a more detailed assessment of specific morphological changes in the hernia-like nodules caused by treatment with a specific inhibitor of CD28-mediated co-stimulation of T-cells.

[0082] 48 female Sprague-Dawley rats weighing approximately 225 g are evenly divided into three groups: Control group (Control, n=16); low dose treatment (T-L n=16); high dose treatment (T-H, n=16). The rats are housed with free access to food and water in environmental-enriched cages. All rats undergo the same surgical procedure as described below. The researcher performing the surgery is blinded to treatment groups.

[0083] Anesthesia is induced and maintained by the inhalation of Isoflurane. A single dose of 0.05 mg/kg buprenorphine (Temgesic) is administered subcutaneously before the procedure for per- and postoperative analgesia.

[0084] Following induction of anesthesia, a skin incision of approximately 5 cm is made in the midline over the spinous processes of the lumbar and caudal spine. An incision is made in the thoracolumbar fascia from level L3-L6. The spinal muscles on both sides on level L4/5 are dissected as well as the supra- and interspinous ligaments to expose the laminae and the interlaminar space at L4/5. A small laminotomy is performed on the caudal aspect of L4 using a surgical micro drill. A 23 g needle is then used to carefully incise the exposed dural sac. The needle is introduced into the cauda equina and the underlying L4/5 disc identified by probing with the needle. The disc is punctured and a small amount of air (0.2 ml) is injected into the disc to facilitate leakage of nucleus pulposus. The needle is then removed, the thoracolumbar fascia sutured and the skin closed with metal clips.

[0085] Therapeutic compounds are administered by intraperitoneal injections once a week.

[0086] To assess the size of the disc nodules following disc puncture, MRI images are obtained with a 7T MRI system (Bruker BioSpec). Measurements are performed week 24 and 8 after surgery. The rats are anesthetized by inhalation of isoflurane throughout the MRI measurements. During the measurements, breathing frequency is monitored and the body temperature maintained by placing the rat on a heated pad.

[0087] A sagittal T2-weighted 3D RARE sequence with isotropic voxels of 150 m is used. The volume of the nodule is measured by manually defining a region of interest (ROI) of the disc aggrandizement dorsal to a longitudinal line between the most dorsal aspects of the adjacent vertebrae on each image where the disc can be identified. The number of voxels within the ROI is assessed three times for each image. The average number of voxels for each sagittal section of the disc are added and then multiplied by the voxel volume (0.003375 mm.sup.3), resulting in the total nodule size expressed in mm.sup.3. In addition to this, a subjective assessment of the nodule size is also performed by the observer for each MM. The researcher collecting the data is blinded to treatment groups.

[0088] Upon termination of the study, a macroscopic analysis is performed in addition to the MRI measurements. After the final MRI measurement 8 weeks after surgery the rats are euthanized by incision of the heart while still anesthetized by inhalation of isoflurane, causing the rat to quickly bleed to death. The lumbar spine is dissected by removing the spinal muscles and the vertebral arches of L3 through L6, exposing the underlying intervertebral disc. A macroscopic assessment of the nodule size is performed through the surgical microscope. The assessments of the nodule size were performed according to the description in Table 1.

[0089] Some of the animals from all groups are terminated at various time points previous to week 8 to allow for harvest of the punctured intervertebral discs for assessments with histology and immunohistochemistry.

[0090] The results from this study support that targeted inhibition of T cell activation, more specifically inhibition of CD28-mediated co-stimulation, can be used to treat disc herniation by inducing and/or expediting resorption of the hernia as well as reducing inflammation in the hernia.

Example 4: Treatment of Disc Herniation with an Inhibitor of CD28-Mediated Co-Stimulation of T-Cells, Patient Scenario

[0091] This example is a fictional case report meant to illustrate how treatment with an inhibitor of CD28-mediated co-stimulation of T-cells is intended to be used and hypothesized to work in the treatment of disc herniation in a clinical setting.

[0092] A 40-year-old woman presents to the primary care facility with a 6 week history of low back pain and sciatic pain in her left leg. She has experienced back pain on occasion before but is otherwise healthy without any medication. She has now tried physiotherapy with limited symptom relief and is still unable to work due to the severity of her symptoms. The primary care physician performs a physical examination and finds the patient has reduced sensory function in her left leg corresponding to the dermatome of 51. The left Achilles reflex is weaker than the right and she has reduced strength in plantar flexion of the left ankle. A left-sided positive Lasgue's sign is noted with pain distribution corresponding to the 51 nerve root.

[0093] The primary care physician suspects a lumbar disc herniation with radiculopathy of the left 51 nerve root. A referral is written for MRI of the lumbar spine to confirm the diagnosis. The patient is also given a prescription for acetaminophen and a non-steroidal anti-inflammatory drug for symptom relief.

[0094] Four weeks later, the patient has undergone MRI and is back at the primary care facility for reassessment. She has not experienced any improvement in her symptoms and uses the prescribed analgesics daily. MRI has confirmed a L5/S1 left-sided disc extrusion with deformation of the left 51 nerve root. A referral is now sent to the spine clinic where the patient is accepted for a new outpatient reassessment in three weeks.

[0095] At the spine clinic, the patient confirms the absence of improvement the past two months. Through physical assessment and assessment of MR images the physician at the spine clinic concludes that the patient has a L5/S1 disc extrusion with symptoms corresponding to this finding. Since there is no spontaneous improvement in symptoms and after ruling out contraindications, the patient is offered and accepts treatment with a drug that inhibits the CD28-mediated co-stimulation of T-cells. A nurse at the spine clinic shows the patient how to self-administer the drug, for example subcutaneously.

[0096] The patient picks up the drug from the local pharmacy and commence treatment at home by administering the drug according to instruction, for example once every one or two weeks. 2-3 weeks after starting treatment the patient starts experiencing improvements in her symptoms, and after 6-8 weeks the symptoms are vastly improved. After a total treatment time of for example 12 weeks, treatment is ended.

[0097] On follow-up MRI 6 months after starting treatment there is still a visible extrusion but it has markedly decrease in size and the adjacent nerve root is no longer affected. The patient is still free from symptoms.

TABLE-US-00001 TABLE 1 Variables collected during macroscopic analysis. Inflammation Degree of hyperemia 0 = No hyperemia of the disc/nodule (+) = Slight hyperemia + = Clear hyperemia ++ = Pronounced hyperemia Healing Healing of puncture 0 = No healing site (+) = Gel-like healing + = Elastic N/A = Not applicable Nodule size Size of lesion over 0 = No nodule the puncture site (+) = Small nodule + = Clear nodule ++ = Pronounced nodule Nodule Consistency of (+) = Gel-like healing consistency nodule as determined + = Elastic by probing ++ = Hard N/A = Not applicable Osteophytes Osteophytes on 0 = No osteophytes adjacent vertebrae (+) = Tendency of osteophytes + = Clear osteophytes ++ = Pronounced osteophytes Infection Pus noted during 0 = Not present dissection + = Present

TABLE-US-00002 TABLE 2 Results from macroscopic analysis. Variables as defined in Table 1. Nodule Nodule Rat Inflammation Healing size consistency Osteophytes DP (Control) 1 (+) + ++ (+) 0 2 0 + 0 (+) 3 (+) + (+) (+) (+) 4 (+) + ++ (+) + 5 + + ++ (+) 0 6 (+) + + (+) (+) 7 (+) + + (+) + 8 (+) + + + + 9 (+) + ++ + ++ 10 (+) + ++ (+) 0 DP + abatacept 11 0 0 0 0 12 0 0 0 (+) 13 0 + 0 (+) 14 0 + 0 (+) 15 (+) + 0 0 16 (+) + (+) (+) (+) 17 0 + 0 0 18 0 + (+) (+) (+) 19 0 + 0 0 20 0 + 0 0

TABLE-US-00003 TABLE 3 Outline of study for assessment of nodule size. 0 1 2 3 4 5 6 7 Group Week Control Saline Saline Saline Saline Saline Saline Saline Saline Treatm. Abat. Abat. Abat. Abat. Abat. Abat. Abat. Abat. start day 0 10 mg/kg 10 mg/kg 10 mg/kg 10 mg/kg 10 mg/kg 10 mg/kg 10 mg/kg 10 mg/kg Treatm. Saline Saline Abat. Abat. Abat. Abat. Abat. Abat. start day 10 mg/kg 10 mg/kg 10 mg/kg 10 mg/kg 10 mg/kg 10 mg/kg 14 Abat. = Abatacept.

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