The Effects of Interferon Beta Combined With Vitamin D on Relapsing Remitting Multiple Sclerosis Patients

Detailed Description of Study Evaluations

Adherence to treatment evaluation:

Patients will be asked to bring the empty packages of the IFN beta and Vitamin D that were used during the study.

Flu like symptoms and Injection site reactions evaluation:

Data regarding FLS and ISRs will be collected by means of a telephone interview every other week. The extent of FLS on the day of injection will be evaluated using a self-rating scale. A score from 0 (not at all) to 5 (very strong), will be given to each FLS component, i.e.: myalgia, chills, headache, malaise and fatigue, exacerbation of neurologic symptoms, sweating and difficulty to function compared to the time before injection. Patient will also record their oral temperature upon awakening as well as at their discretion. All patients will be provided with the same thermometer. Participants will be asked to report the frequency of Ibuprofen or Paracetamol use. and will be recommended not to exceed 800mg of Ibuprofen or 1000mg of Paracetamol per day, unless the symptoms are unbearable.

Patients will be inquired about the subjective degree of injection site pain for the first injections of every other week, by means of a 100 mm visual analogue scale (VAS scale). Patients will be seen at the clinic at 3 months intervals and in that visit the injection site will be inspected by the evaluating physician/ nurse and will be graded on a 0 to 3 scale: 0-none, 1- pain, itching or erythema, 2- swelling with inflammation and 3- ulceration and/or necrosis. Mode of application (manual or automatic injection) as well as needle size will be also recorded.

Patients will be instructed to refrain from reporting FLS and ISRs on the occasion of inter-current febrile illness or during and 4 weeks after corticosteroids treatment. In that case, patients will postpone the report by a week.

MS clinical parameters:

Patients will be clinically evaluated at enrollment and at 6 and 12 months for disease progression, by means of the EDSS score . The evaluating neurologist will be blinded to the patient's treatment.

Relapses, characterized by emergence of new symptoms or the worsening of existing ones for more than 24 hours in the absence of fever or infection ( McDonald et al., 2001) and severe enough to warrant corticosteroids treatment will be recorded.

Health related quality of life will be also evaluated at baseline and at the completion of the study, by means of a MS-specific validated questionnaire - FAMS (Functional Assessment of MS) (Miller A, 2006). (Attached)

Laboratory evaluation:

The following measurements will be obtained at the beginning of the study for the purpose of excluding patients with baseline impairment of mineral metabolism and at the end of the study to evaluate the resulting changes in mineral homeostasis consequent to vitamin D supplementation: blood levels of 25-OH- vitamin D, PTH, Magnesium, Calcium, Albumin, Creatinine, Urea, Phosphor, Alkaline phosphatase and 1-25- di-OH-D .

The following measurements will be assessed after 3, 6 and 12 months from study start for the purpose of 25-OH-D level monitoring and safety: Calcium, Phosphor, 25-OH-D , Albumin and creatinine. Laboratory evaluation required with IFN-beta safety will include: Complete blood count at baseline and after 3, 6, and 12 months from study start ; liver function tests at baseline and every 3 months, thyroid function tests at baseline, 6 months and by the end of the study or upon the emergence of symptoms suggestive of thyroid dysfunction.

Immunological markers:

Two venous blood samples will be drawn from a number of participants at Baseline and after 3 months of vitamin D supplementation . The first in the morning of IFN beta injection day and the second in the morning of the day after the injection. The percent rise after IFN beta injection of IL-6 and TRAIL will be assessed by commercial ELISA kits.

On visits baseline, 3 and 12 month, a venous blood portion will be stored at -80 oC within 30 min- 4 hour of blood withdrawal for MS related cytokines and proteins evaluation.

On visits baseline and 6 months another portion will be collected for immediately immune cell subset evaluation. PBMCs will isolated within 4 hour by Ficoll gradient centrifugation, stained with specific antibodies characterizing various immune cell subsets, and subset proportion determined by flow cytometry analysis.

Blood sampling will be postponed in case of a known condition that may possibly influence the expression of the studied cytokines. Namely, suspicion of a new or ongoing relapse within 30 days before or during the date of blood sampling; Clinical or paraclinical signs of acute infectious disease 48 hrs prior to blood sampling (leukocyte count > 10,000/mcl and /or CRP >2 mg/dl); Clinical signs of allergy within 7 days before participation; corticosteroids within 30 days before participation.

Melatonin:

Urine will be collected during 12-hour nighttime (20:00-08:00) at baseline, 3 months and by the end of the study. Levels of the melatonin metabolite (6-sulphatoxy-melatonin) will be assessed using a highly specific ELISA assay.