Cytokine biomarkers as predictive biomarkers of clinical response for glatiramer acetate

Abstract

A method for treating a human subject afflicted with multiple sclerosis or a single clinical attack consistent with multiple sclerosis with a pharmaceutical composition comprising glatiramer acetate and a pharmaceutically acceptable carrier, comprising the steps of determining whether the human subject is a glatiramer acetate responder by evaluating a biomarker selected from the group consisting of IL-17 concentration, TNF- concentration, IL-2 concentration and IFN- concentration, or a combination thereof, in the blood of the human subject and administering the pharmaceutical composition comprising glatiramer acetate and a pharmaceutically acceptable carrier to the human subject only if the human subject is identified as a glatiramer acetate responder.

Claims

1. A method for treating a human subject afflicted with multiple sclerosis or a single clinical attack consistent with multiple sclerosis with a pharmaceutical composition comprising glatiramer acetate and a pharmaceutically acceptable carrier, comprising the steps of: a) determining whether the human subject is a glatiramer acetate responder by evaluating IFN- concentration in a supernatant of PBMCs derived from the human subject's blood, which human subject can be identified as a glatiramer acetate responder by steps comprising, (i) purifying PBMCs from the subject's blood and then cryopreserving the PBMCs, (ii) thawing and then culturing the cryopreserved PBMCs overnight in culture medium supplemented with 5% human serum, (iii) stimulating about 40,000 of said cultured PBMCs with 1 mg/ml PMA and 5 mg/ml ionomycin in a final volume of 200 microliters for 6 hours, and (iv) identifying the human subject as a glatiramer acetate responder if IFN- concentration of a supernatant of PBMCs after step (iii) is greater than or equal to 6000 pg/ml; and b) administering the pharmaceutical composition comprising glatiramer acetate and a pharmaceutically acceptable carrier to the human subject only if the human subject is identified as a glatiramer acetate responder, wherein the human subject is a naive patient or wherein the human subject has been previously administered a multiple sclerosis drug other than glatiramer acetate.

2. The method of claim 1, wherein administering the pharmaceutical composition comprising glatiramer acetate and a pharmaceutically acceptable carrier comprises administering to the human subject three subcutaneous injections of the pharmaceutical composition over a period of seven days with at least one day between every subcutaneous injection.

3. The method of claim 1, wherein the pharmaceutical composition is a unit dose of a 0.5 ml aqueous solution comprising 20 mg of glatiramer acetate.

4. The method of claim 1, wherein if the human subject is identified as a glatiramer acetate responder, the human subject is thereafter administered the pharmaceutical composition comprising glatiramer acetate and a pharmaceutically acceptable carrier as monotherapy.

5. The method of claim 1, wherein if the human subject is not identified as a glatiramer acetate responder, the human subject is thereafter administered a multiple sclerosis drug which is not glatiramer acetate.

6. The method of claim 1, wherein the human subject is a naive patient.

7. The method of claim 1, further comprising in step (iv) identifying the human subject as a glatiramer acetate responder based on IL-17 concentration, TNF- concentration, IL-2 concentration, or a combination thereof, of a supernatant of the PBMCs after step (iii).

8. The method of claim 1, comprising in step (iv) identifying the human subject as a glatiramer acetate responder based on IFN- concentration, IL-17 concentration, TNF- concentration and IL-2 concentration of a supernatant of the PBMCs after step (iii).

9. A method of predicting clinical responsiveness to glatiramer acetate therapy in a human subject afflicted with multiple sclerosis or a single clinical attack consistent with multiple sclerosis, the method comprising evaluating IFN- concentration in a supernatant of PBMCs derived from the human subject's blood, which human subject can be identified as a glatiramer acetate responder by steps comprising, (i) purifying PBMCs from the subject's blood and then cryopreserving the PBMCs, (ii) thawing and then culturing the cryopreserved PBMCs overnight in culture medium supplemented with 5% human serum, (iii) stimulating about 40,000 of said cultured PBMCs with 1 mg/ml PMA and 5 mg/ml ionomycin in a final volume of 200 microliters for 6 hours, and (iv) identifying the human subject as a glatiramer acetate responder if IFN- concentration of a supernatant of PBMCs after step (iii) is greater than or equal to 6000pg/ml, to thereby predict clinical responsiveness to glatiramer acetate, wherein the human subject is a naive patient or wherein the human subject has been previously administered a multiple sclerosis drug other than glatiramer acetate.

10. The method of claim 9, wherein the glatiramer acetate therapy comprises administering to the human subject three subcutaneous injections of a pharmaceutical composition comprising glatiramer acetate and a pharmaceutically acceptable carrier over a period of seven days with at least one day between every subcutaneous injection.

11. The method of claim 9, further comprising in step (iv) identifying the human subject as a glatiramer acetate responder based on IL-17 concentration, TNF- concentration, IL-2 concentration, or a combination thereof, of a supernatant of the PBMCs after step (iii).

12. The method of claim 9, comprising in step (iv) identifying the human subject as a glatiramer acetate responder based on IFN- concentration, IL-17 concentration, TNF- concentration and IL-2 concentration of a supernatant of the PBMCs after step (iii).

13. The method of claim 9, wherein the human subject is a naive patient.

14. A method for treating a human subject afflicted with multiple sclerosis or a single clinical attack consistent with multiple sclerosis comprising the steps of: a) administering to the human subject a therapeutic amount of a pharmaceutical composition comprising glatiramer acetate and a pharmaceutically acceptable carrier, wherein the human subject is a naive patient or wherein the human subject has been previously administered a multiple sclerosis drug other than glatiramer acetate; b) evaluating IFN- concentration in a supernatant of PBMCs derived from the human subject's blood, which human subject can be identified as a glatiramer acetate responder by steps comprising, (i) purifying PBMCs from the subject's blood and then cryopreserving the PBMCs, (ii) thawing and then culturing the cryopreserved PBMCs overnight in culture medium supplemented with 5% human serum, (iii) stimulating about 40,000 of said cultured PBMCs with 1 mg/ml PMA and 5 mg/ml ionomycin in a final volume of 200 microliters for 6 hours, and (iv) identifying the human subject as a glatiramer acetate responder if IFN- concentration of a supernatant of PBMCs after step (iii) is greater than or equal to 6000pg/ml; and c) continuing administration of the pharmaceutical composition if the human subject is identified as a glatiramer acetate responder, or modifying the administration of the pharmaceutical composition to the human subject if the human subject is not identified as a glatiramer acetate responder.

15. The method of claim 14, wherein administering to the human subject a therapeutic amount of a pharmaceutical composition comprising glatiramer acetate and a pharmaceutically acceptable carrier comprises administering to the human subject three subcutaneous injections of the pharmaceutical composition over a period of seven days with at least one day between every subcutaneous injection.

16. The method of claim 14, wherein the IFN- concentration is observed at 2 months after the first administration of glatiramer acetate.

17. The method of claim 14, further comprising in step (iv) identifying the human subject as a glatiramer acetate responder based on IL-17 concentration, TNF- concentration, IL-2 concentration, or a combination thereof, of a supernatant of the PBMCs after step (iii).

18. The method of claim 14, comprising in step (iv) identifying the human subject as a glatiramer acetate responder based on IFN- concentration, IL-17 concentration, TNF- concentration and IL-2 concentration of a supernatant of the PBMCs after step (iii).

19. The method of claim 14, wherein the human subject is a naive patient.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1A: Time line showing responders' EDSS score with GA treatment.

(2) FIG. 1B: Time line showing non-responders' EDSS score with GA treatment.

(3) FIG. 2A: Cytokine levels secreted by the Peripheral blood mononuclear cells (PBMCs) of responders at baseline and after 2 months of GA treatment.

(4) FIG. 2B: Cytokine levels secreted by PBMCs of non-responders at baseline and after 2 months of GA treatment.

DETAILED DESCRIPTION OF THE INVENTION

(5) This invention provides a method for treating a human subject afflicted with multiple sclerosis or a single clinical attack consistent with multiple sclerosis with a pharmaceutical composition comprising glatiramer acetate and a pharmaceutically acceptable carrier, comprising the steps of determining whether the human subject is a glatiramer acetate responder by evaluating a biomarker selected from the group consisting of IL-17 concentration, TNF- concentration, IL-2 concentration and IFN- concentration, or a combination thereof, in the blood of the human subject and administering the pharmaceutical composition comprising glatiramer acetate and a pharmaceutically acceptable carrier to the human subject only if the human subject is identified as a glatiramer acetate responder.

(6) In an embodiment, administering the pharmaceutical composition comprising glatiramer acetate and a pharmaceutically acceptable carrier comprises administering to the human subject three subcutaneous injections of the pharmaceutical composition over a period of seven days with at least one day between every subcutaneous injection.

(7) In an embodiment, the pharmaceutical composition is a unit dose of a 0.5 ml aqueous solution comprising 20 mg of glatiramer acetate.

(8) In an embodiment, the IL-17 concentration, TNF- concentration, IL-2 concentration and IFN- concentration, or the combination thereof is a PBMC supernatant concentration.

(9) In an embodiment, the IL-17 concentration, TNF- concentration, IL-2 concentration and IFN- concentration, or the combination thereof is observed at pretreatment.

(10) In an embodiment, the IL-17 concentration, INF- concentration, IL-2 concentration and INF- concentration, or the combination thereof is observed at 2 months after the first administration of glatiramer acetate.

(11) In an embodiment, if the human subject is identified as a glatiramer acetate responder, the human subject is thereafter administered the pharmaceutical composition comprising glatiramer acetate and a pharmaceutically acceptable carrier as monotherapy.

(12) In an embodiment, if the human subject is identified as a glatiramer acetate responder, the human subject is thereafter administered a pharmaceutical composition comprising glatiramer acetate and a pharmaceutically acceptable carrier, and the human subject is also thereafter administered another multiple sclerosis drug which is not glatiramer acetate. In a further embodiment, the multiple sclerosis drug is selected from Interferon, Mitoxantrone and Natalizumab.

(13) In an embodiment, if the human subject is not identified as a glatiramer acetate responder, the human subject is thereafter administered a multiple sclerosis drug which is not glatiramer acetate.

(14) In an embodiment, if the human subject is not identified as a glatiramer acetate responder, the human subject is thereafter administered a multiple sclerosis drug which is not glatiramer acetate, and the human subject is not thereafter administered glatiramer acetate.

(15) In an embodiment, the multiple sclerosis drug is selected from Interferon, Mitoxantrone and Natalizumab.

(16) In an embodiment, the biomarker is IL-17 concentration.

(17) In an embodiment, the biomarker is IL-17(A) concentration. In a further embodiment, an IL-17 concentration or an IL-17(A) concentration greater than or equal to 120 pg/ml is associated with a human subject identified as a glatiramer acetate responder.

(18) In an embodiment, the biomarker is TNF- concentration. In a further embodiment, a TNF- concentration greater than or equal to 20000 pg/ml is associated with a human subject identified as a glatiramer acetate responder.

(19) In an embodiment, the biomarker is IFN- concentration. In a further embodiment, an IFN- concentration greater than or equal to 6000 pg/ml is associated with a human subject identified as a glatiramer acetate responder.

(20) In an embodiment, the biomarker is IL-2 concentration. In a further embodiment, an IL-2 concentration greater than or equal to 30000 pg/ml is associated with a human subject identified as a glatiramer acetate responder.

(21) In an embodiment, the human subject is a naive patient.

(22) In an embodiment, the human subject has been previously administered a multiple sclerosis drug other than glatiramer acetate. In a further embodiment, the previously administered multiple sclerosis drug is selected from Interferon, Mitoxantrone and Natalizumab.

(23) This invention also provides a method of predicting clinical responsiveness to glatiramer acetate therapy in a human subject afflicted with multiple sclerosis or a single clinical attack consistent with multiple sclerosis, the method comprising evaluating a biomarker selected from the group consisting of IL-concentration, INF- concentration, IL-2 concentration and IFN- concentration, or a combination thereof, in the blood of the human subject, to thereby predict clinical responsiveness to glatiramer acetate.

(24) In an embodiment, the glatiramer acetate therapy comprises administering to the human subject three subcutaneous injections of a pharmaceutical composition comprising glatiramer acetate and a pharmaceutically acceptable carrier over a period of seven days with at least one day between every subcutaneous injection. In a further embodiment, the pharmaceutical composition is a unit dose of a 0.5 ml aqueous solution comprising 20 mg of glatiramer acetate.

(25) In an embodiment, the IL-17 concentration, INF- concentration, IL-2 concentration and IFN- concentration, or the combination thereof is a PBMC supernatant concentration.

(26) In an embodiment, the IL-17 concentration, INF- concentration, IL-2 concentration and IFN- concentration, or the combination thereof is observed at pretreatment.

(27) In an embodiment, the IL-17 concentration, INF- concentration, IL-2 concentration and INF- concentration, or the combination thereof is observed at 2 months after the first administration of glatiramer acetate.

(28) In an embodiment, if the human subject is identified as a glatiramer acetate responder, the human subject is thereafter administered the pharmaceutical composition comprising glatiramer acetate and a pharmaceutically acceptable carrier as monotherapy.

(29) In an embodiment, if the human subject is identified as a glatiramer acetate responder, the human subject is thereafter administered a pharmaceutical composition comprising glatiramer acetate and a pharmaceutically acceptable carrier, and the human subject is also thereafter administered another multiple sclerosis drug which is not glatiramer acetate. In a further embodiment, the multiple sclerosis drug is selected from Interferon, Mitoxantrone and Natalizumab.

(30) In an embodiment, if the human subject is not identified as a glatiramer acetate responder, the human subject is thereafter administered a multiple sclerosis drug which is not glatiramer acetate.

(31) In an embodiment, if the human subject is not identified as a glatiramer acetate responder, the human subject is thereafter administered a multiple sclerosis drug which is not glatiramer acetate, and the human subject is not thereafter administered glatiramer acetate.

(32) In an embodiment, the multiple sclerosis drug is selected from Interferon, Mitoxantrone and Natalizumab.

(33) In an embodiment, the biomarker is IL-17 concentration.

(34) In an embodiment, the biomarker is IL-17(A) concentration. In a further embodiment, an IL-17 concentration or an IL-17(A) concentration greater than or equal to 120 pg/ml is associated with a human subject identified as a glatiramer acetate responder.

(35) In an embodiment, the biomarker is TNF- concentration. In a further embodiment, a TNF- concentration greater than or equal to 20000 pg/ml is associated with a human subject identified as a glatiramer acetate responder.

(36) In an embodiment, the biomarker is IFN- concentration. In a further embodiment, an IFN- concentration greater than or equal to 6000 pg/ml is associated with a human subject identified as a glatiramer acetate responder.

(37) In an embodiment, the biomarker is IL-2 concentration. In a further embodiment, an IL-2 concentration greater than or equal to 30000 pg/ml is associated with a human subject identified as a glatiramer acetate responder.

(38) In an embodiment, the human subject is a naive patient.

(39) In an embodiment, the human subject has been previously administered a multiple sclerosis drug other than glatiramer acetate. In a further embodiment, the previously administered multiple sclerosis drug is selected from Interferon, Mitoxantrone and Natalizumab.

(40) This invention also provides a method for treating a human subject afflicted with multiple sclerosis or a single clinical attack consistent with multiple sclerosis comprising the steps of administering to the human subject a therapeutic amount of a pharmaceutical composition comprising glatiramer acetate and a pharmaceutically acceptable carrier, determining whether the human subject is a glatiramer acetate responder by evaluating a biomarker selected from the group consisting of IL-17 concentration, TNF- concentration, IL-2 concentration and IFN- concentration, or a combination thereof, in the blood of the human subject, and continuing administration of the pharmaceutical composition if the human subject is identified as a glatiramer acetate responder, or modifying the administration of the pharmaceutical composition to the human subject if the human subject is not identified as a glatiramer acetate responder.

(41) In an embodiment, administering to the human subject a therapeutic amount of a pharmaceutical composition comprising glatiramer acetate and a pharmaceutically acceptable carrier comprises administering to the human subject three subcutaneous injections of the pharmaceutical composition over a period of seven days with at least one day between every subcutaneous injection.

(42) In an embodiment, the pharmaceutical composition is a unit dose of a 0.5 ml aqeuous solution comprising 20 mg of glatiramer acetate.

(43) In an embodiment, the IL-17 concentration, INF- concentration, IL-2 concentration and IFN- concentration, or the combination thereof is a PBMC supernatant concentration.

(44) In an embodiment, the IL-17 concentration, INF- concentration, IL-2 concentration and IFN- concentration, or the combination thereof is observed at pretreatment.

(45) In an embodiment, the IL-17 concentration, INF- concentration, IL-2 concentration and INF- concentration, or the combination thereof is observed at 2 months after the first administration of glatiramer acetate.

(46) In an embodiment, if the human subject is identified as a glatiramer acetate responder, the human subject is thereafter administered the pharmaceutical composition comprising glatiramer acetate and a pharmaceutically acceptable carrier as monotherapy.

(47) In an embodiment, if the human subject is identified as a glatiramer acetate responder, the human subject is thereafter administered a pharmaceutical composition comprising glatiramer acetate and a pharmaceutically acceptable carrier, and the human subject is also thereafter administered another multiple sclerosis drug which is not glatiramer acetate. In a further embodiment, the multiple sclerosis drug is selected from Interferon, Mitoxantrone and Natalizumab.

(48) In an embodiment, if the human subject is not identified as a glatiramer acetate responder, the human subject is thereafter administered a multiple sclerosis drug which is not glatiramer acetate.

(49) In an embodiment, if the human subject is not identified as a glatiramer acetate responder, the human subject is thereafter administered a multiple sclerosis drug which is not glatiramer acetate, and the human subject is not thereafter administered glatiramer acetate.

(50) In an embodiment, the multiple sclerosis drug is selected from Interferon, Mitoxantrone and Natalizumab.

(51) In an embodiment, the biomarker is IL-17 concentration.

(52) In an embodiment, the biomarker is IL-17(A) concentration. In a further embodiment, an IL-17 concentration or an IL-17(A) concentration greater than or equal to 120 pg/ml is associated with a human subject identified as a glatiramer acetate responder.

(53) In an embodiment, the biomarker is TNF- concentration. In a further embodiment, a TNF- concentration greater than or equal to 20000 pg/ml is associated with a human subject identified as a glatiramer acetate responder.

(54) In an embodiment, the biomarker is IFN- concentration. In a further embodiment, an IFN- concentration greater than or equal to 6000 pg/ml is associated with a human subject identified as a glatiramer acetate responder.

(55) In an embodiment, the biomarker is IL-2 concentration. In a further embodiment, an IL-2 concentration greater than or equal to 30000 pg/ml is associated with a human subject identified as a glatiramer acetate responder.

(56) In an embodiment, the human subject is a naive patient.

(57) In an embodiment, the human subject has been previously administered a multiple sclerosis drug other than glatiramer acetate. In a further embodiment, the previously administered multiple sclerosis drug is selected from Interferon, Mitoxantrone and Natalizumab.

DEFINITIONS

(58) Forms of Multiple Sclerosis:

(59) There are five distinct disease stages and/or types of MS: 1) benign multiple sclerosis; 2) relapsing-remitting multiple sclerosis (RRMS); 3) secondary progressive multiple sclerosis (SPMS); 4) progressive relapsing multiple sclerosis (PRMS); and 5) primary progressive multiple sclerosis (PPMS).

(60) Benign multiple sclerosis is a retrospective diagnosis which is characterized by 1-2 exacerbations with complete recovery, no lasting disability and no disease progression for 10-15 years after the initial onset. Benign multiple sclerosis may, however, progress into other forms of multiple sclerosis.

(61) Patients suffering from RRMS experience sporadic exacerbations or relapses, as well as periods of remission. Lesions and evidence of axonal loss may or may not be visible on MRI for patients with RRMS.

(62) SPMS may evolve from RRMS. Patients afflicted with SPMS have relapses, a diminishing degree of recovery during remissions, less frequent remissions and more pronounced neurological deficits than RRMS patients. Enlarged ventricles, which are markers for atrophy of the corpus callosum, midline center and spinal cord, are visible on MRI of patients with SPMS.

(63) PPMS is characterized by a steady progression of increasing neurological deficits without distinct attacks or remissions. Cerebral lesions, diffuse spinal cord damage and evidence of axonal loss are evident on the MRI of patients with PPMS. PPMS has periods of acute exacerbations while proceeding along a course of increasing neurological deficits without remissions. Lesions are evident on MRI of patients suffering from PRMS.(28)

(64) A clinically isolated syndrome (CIS) is a single monosymptomatic attack compatible with MS, such as optic neuritis, brain stem symptoms, and partial myelitis. Patients with CIS that experience a second clinical attack are generally considered to have clinically definite multiple sclerosis (CDMS). Over 80 percent of patients with a CIS and MRI lesions go on to develop MS, while approximately 20 percent have a self-limited process.(29,30) Patients who experience a single clinical attack consistent with MS may have at least one lesion consistent with multiple sclerosis prior to the development of clinically definite multiple sclerosis.

(65) Multiple sclerosis may present with optic neuritis, blurring of vision, diplopia, involuntary rapid eye movement, blindness, loss of balance, tremors, ataxia, vertigo, clumsiness of a limb, lack of co-ordination, weakness of one or more extremity, altered muscle tone, muscle stiffness, spasms, tingling, paraesthesia, burning sensations, muscle pains, facial pain, trigeminal neuralgia, stabbing sharp pains, burning tingling pain, slowing of speech, slurring of words, changes in rhythm of speech, dysphagia, fatigue, bladder problems (including urgency, frequency, incomplete emptying and incontinence), bowel problems (including constipation and loss of bowel control), impotence, diminished sexual arousal, loss of sensation, sensitivity to heat, loss of short term memory, loss of concentration, or loss of judgment or reasoning.

(66) Relapsing Form of Multiple Sclerosis:

(67) The term relapsing MS includes: 1) patients with RRMS; 2) patients with SPMS and superimposed relapses; and 3) patients with CIS who show lesion dissemination on subsequent MRI scans according to McDonald's criteria.

(68) As used herein, relapsing forms of multiple sclerosis include: Relapsing-remitting multiple sclerosis (RRMS), characterized by unpredictable acute episodes of neurological dysfunction (relapses), followed by variable recovery and periods of clinical stability;

(69) Secondary Progressive MS (SPMS), wherein patients having RRMS develop sustained deterioration with or without relapses superimposed; and

(70) Primary progressive-relapsing multiple sclerosis (PPRMS) or progressive-relapsing multiple sclerosis (PRMS), an uncommon form wherein patients developing a progressive deterioration from the beginning can also develop relapses later on.

(71) Kurtzke Expanded Disability Status Scale (EDSS):

(72) The Kurtzke Expanded Disability Status Scale (EDSS) is a method of quantifying disability in multiple sclerosis. The EDSS replaced the previous Disability Status Scales which used to bunch people with MS in the lower brackets. The EDSS quantifies disability in eight Functional Systems (FS) and allows neurologists to assign a Functional System Score (FSS) in each of these. The Functional Systems are: pyramidal, cerebellar, brainstem, sensory, bowel and bladder, visual & cerebral (according to www.mult-sclerosis.org/expandeddisabil itystatusscale).

(73) Clinical Relapse:

(74) A clinical relapse, which may also be used herein as relapse, confirmed relapse, or clinically defined relapse, is defined as the appearance of one or more new neurological abnormalities or the reappearance of one or more previously observed neurological abnormalities.

(75) This change in clinical state must last at least 48 hours and be immediately preceded by a relatively stable or improving neurological state of at least 30 days. This criterion is different from the clinical definition of exacerbation at least 24 hours duration of symptoms, (31) as detailed in the section relapse evalution.

(76) An event is counted as a relapse only when the subject's symptoms are accompanied by observed objective neurological changes, consistent with: a) an increase of at least 1.00 in the EDSS score or one grade in the score of two or more of the seven FS (32); or, b) two grades in the score of one of FS as compared to the previous evaluation.

(77) The subject must not be undergoing any acute metabolic changes such as fever or other medical abnormality. A change in bowel/bladder function or in cognitive function must not be entirely responsible for the changes in EDSS or FS scores.

(78) As used herein, in the blood of the subject is represented by serum and also the supernatant of PBMCs derived from the subject's blood.

(79) As used herein, the supernatant refers to supernatants collected from Peripheral blood mononuclear cells (PBMCs) purified from subject blood samples, and stimulated as described in the methods hereinbelow. The stimulation may be performed in either freshly isolated PBMCs or in cryopreserved cells after thawing.

(80) As used herein, concentration observed at a certain time-point refers to a concentration measured in the supernatant of PBMC derived from the subject's blood at that certain time point. The concentration may be measured in freshly isolated cells or in cryopreserved cells after thawing.

(81) As used herein, pretreatment refers to any time point after diagnosis with MS or CIS and before beginning of treatment with a composition comprising GA.

(82) As used herein, a multiple sclerosis drug is a drug or an agent intended to treat clinically defined MS, CIS, any form of neurodegenerative or demyelinating diseases, or symptoms of any of the above mentioned diseases. Multiple sclerosis drugs may include but are not limited to antibodies, immunosuppressants, anti-inflammatory agents, immunomodulators, cytokines, cytotoxic agents and steroids and may include approved drugs, drugs in clinical trial, or alternative treatments, intended to treat clinically defined MS, CIS or any form of neurodegenerative or demyelinating diseases. Multiple sclerosis drugs include but are not limited to Interferon and its derivatives (including BETASERON, AVONEX and REBIF), Mitoxantrone and Natalizumab. Agents approved or in-trial for the treatment of other autoimmune diseases, but used in a MS or CIS patient to treat MS or CIS are also defined as multiple sclerosis drugs.

(83) As used herein, a naive patient is a subject that has not been treated with any multiple sclerosis drugs as defined in the former paragraph.

(84) Experimental Details

Example

Evaluating Cytokine Levels in Patients Classified as Responders or Non-Responders to GA

(85) Methods

(86) Subjects and Cells:

(87) Relapsing-remitting multiple sclerosis patients (n=12) were treated with either 20 mg GA or 40 mg GA daily in the Teva FORTE clinical trial(www.medicalnewstoday.com/articles/48863.php). Whole blood was taken from patients at three time points including baseline (baseline, month 2 and month 6). Peripheral blood mononuclear cells (PBMCs) were cryopreserved at baseline, month 2 and month 6.

(88) The incidence of clinical relapses, and the expanded disability status scale score (EDSS) after 12 months treatment were used to define patients as responders (no clinical relapse during the test period) or non-responders (1 or more clinical relapses as defined hereinbelow). Several patients were withdrawn from drug within the treatment year due to adverse responses and were not included in this analysis.

(89) Multiplex Cytokine Assay

(90) Blood was drawn from patients at baseline, 2 months and 6 months. Peripheral Blood Mononuclear Cells (PBMCs) were purified from the blood using a Ficoll-Hypaque gradient and cryopreserved. Cryopreserved PBMCs from each time point were thawed, rested overnight in AIM V medium supplemented with 5% human serum, and stimulated with PMA (1 mg/ml; SIGMA) and ionomycin (5 mg/ml; SIGMA) for 6 hours (40,000 PBMCs in 200 microliters final volume). Supernatants were removed from stimulated and unstimulated cells and stored at 20 C. until assay with a human 27-plex kit (Bio-Rad Laboratories, Hercules, Calif.). Data was acquired using a Bio-Plex Array Reader and analyzed with Bio-Plex Manager 4 software (Bio-Rad). Graphs were drawn using Prism software (GraphPad Software, Inc.). We performed both 2-plex (IL-17 and IFN) and 27 multiplex (including 27 human cytokines) were used in this comprehensive cytokine analysis. The multiplex data from these two assays were individualized on a per patient basis and presented in FIG. 2.

(91) Relapse Evaluation

(92) A clinical relapse was defined as the appearance of one or more new neurological abnormalities or the reappearance of one or more previously observed neurological abnormalities.

(93) This change in clinical state lasted at least 48 hours and was immediately preceded by a relatively stable or improving neurological state of at least 30 days. The criterion used in the study was different from the clinical definition of exacerbation at least 24 hours duration of symptoms. (31) Since in study exacerbation definition must be supported by an objective neurological evaluation (see next paragraph), a neurological deficit must sustain long enough to eliminate pseudo exacerbations.

(94) An event was counted as a relapse only when the subject's symptoms were accompanied by observed objective neurological changes, consistent with: a) an increase of at least 1.00 in the EDSS score or one grade in the score of two or more of the seven FS (32); or, b) two grades in the score of one of FS as compared to the previous evaluation.

(95) The subject was not undergoing any acute metabolic changes such as fever or other medical abnormality. A change in bowel/bladder function or in cognitive function was not entirely responsible for the changes in EDSS or FS scores.

(96) Subject Evaluation by the Examining Neurologist

(97) A complete neurological assessment was performed at months 1 (screening), 0 (baseline), 3, 6, 9, 12 (end of double-blind phase), 18 and 24 (termination/early discontinuation).

(98) Relapse Determination by the Treating Neurologist

(99) The decision as to whether the neurological change was considered a confirmed relapse was made by the Treating Physician, based on EDSS/FS actual (not converted) scores assessed by the Examining Neurologist.

(100) Follow-up visits to monitor the course of the relapse were made at the Treating Physician's discretion, in addition to the assessment at the next scheduled visit, but the neurological assessments were performed by the Examining Neurologist.

(101) Relapse Evaluation Procedures

(102) Subjects were instructed to telephone their study site within 48 hours should any symptoms suggestive of a relapse appear.

(103) The Examining Neurologist evaluated the subject within 7 days of symptoms onset, conditional upon a symptomatic period of 48 hours. The Treating Neurologist/Physician evaluated the subject once any symptom suggestive of a relapse occurred.

(104) In case of a suggestive relapse during a scheduled or unscheduled visit, the Treating Neurologist/Physician referred the subject to the Examining eurologist/Physician.

(105) Results

(106) These findings demonstrate increased levels of IL-17(A), TNF-alpha, IL-2 and IFN-gamma, at the baseline and 2 months time points, in those individuals who were without clinically defined relapses in the one year trial period post initiation of treatment. To the contrary, we observed substantially lower levels of these same pro-inflammatory cytokines in those who had clinically defined relapses during the trial period (see FIG. 1 and FIG. 2).

(107) Discussion

(108) For this study, PBMC were obtained at baseline, 2 months and 6 months post initiation of treatment with glatiramer acetate. The findings demonstrate increased levels of IL-17(A), IL-2, TNF, and IFN at baseline and 2 months after the beginning of treatment with GA, in those individuals who were without clinically defined relapses in the one year trial period post initiation of treatment. To the contrary, substantially lower levels of these same pro-inflammatory cytokines were found in those who had clinically defined relapses during the trial period as shown in FIG. 1.

(109) Ex vivo assays have been used to monitor the immunological effects of GA in GA-treated MS patients. For example, Hohlfeld et al. reported: (1) a significant reduction of GA-induced PBMC proliferation; (2) a positive IL-4 ELISPOT response mediated predominantly by CD4 cells after stimulation with GA; and (3) an elevated IFN-gamma response partially mediated by CD8 cells after stimulation with high GA concentrations, in GA-treated vs. untreated patients (33).

(110) In the present study, a simple ex vivo assay was used to measure cytokine concentration in the supernatant of PBMCs derived from the blood of RRMS patients. The data suggest that specific cytokine patterns may be associated with the identification of those who will respond to therapy with glatiramer acetate. The trend that is seen may be suggestive of cytokine patterns that could be readily measured and assist in determining GA responsiveness before GA treatment, and at an early time-point after the beginning of GA administration.

REFERENCES

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