Intra-discal Injection of Platelet-rich Plasma (PRP) Enriched With Exosomes in Chronic Low Back Pain
Condition: Chronic Low Back Pain; Degenerative Disc Disease
MeSH terms: Intervertebral Disc Degeneration; Back Pain; Low Back Pain
Study type: Interventional
Phase: Phase 1

Summary: 
      Intervertebral disc pathology accounts for 40% or more cases of chronic LBP. Available
      treatment options do not improve the underlying degenerative condition hence This indicates
      the need for new therapies.

      intradiscal injection of PRP with exosomes derived from blood may potentially transmit some
      of the unique stem cell properties to other stem cells, facilitating stemness maintenance,
      differentiation, self-renewal, and repair.

      controlled, randomized, double-blind placebo clinical trial to compare the safety and
      efficacy of PRP with exosomes in discogenic LBP adminstartion of PRP with exosomes at the
      centre of the nucleus pulposus or equal amount of placebo paradiscal patients wil be
      elvaluated for primary Outcome measures viz: Visual analog scale (VAS), Roland Morris
      Disability Questionnaire (RDQ), SF 36 health questionnaire, Functional rating index and
      Secondary: MRI - disc degeneration grading, T2 quantification, and for any Adverse events
      including of discitis
    

Detailed description: 
      Management of chronic low back pain (LBP) has contributed to its high socioeconomic The
      intervertebral disc accounts for 40% or more cases of chronic LBP. Available treatment
      options do not improve the underlying degenerative condition, although they do resolve its
      symptoms. This indicates the need for new therapies and/or interventions that actually treat
      the underlying causes of discogenic pain. Biological therapies by direct injection of PRP
      into the annulus fibrosus and nucleus pulposus seems an effective and feasible option.

      Exosomes derived from blood may potentially transmit some of the unique stem cell properties
      to other stem cells, facilitating stemness maintenance, differentiation, self-renewal, and
      repair.

      We propose to have a controlled, randomized, double-blind clinical trial to compare the
      safety and efficacy of exosomes in LBP. Informed consent will be obtained from all subjects
      prior to study participation.

      PRP Preparation:

      Blood sample (60 ml) will be centrifuged at 600×g for 10 min to collect plasma fraction. And
      centrifuged at 4000×g for 15 min. Supernatant platelet poor plasma (PPP) will be then
      removed, leaving 1 ml PRP. PPP will be passed through one-micron special indigenous designed
      flush back filter (Alpha Corpuscle, Delhi, India) so that all the platelets present in PPP
      fraction will get trapped in the filter. These trapped platelets will be flushed back with 1
      ml of PPP to retrieve most of the platelets. This will be mixed together and then passed
      through a WBC filter (Terumo Imuguard, USA) to filter off the leukocytes. The final PRP
      concentrate (0.1 ml) will be analysed for total leukocyte & platelet counts. Five samples
      will be selected randomly treated with bovine thrombin reconstituted in 10% calcium chloride
      will be assessed for growth factors [platelet-derived growth factors (PDGF) and vascular
      endothelial growth factors (VEGF)] by ELISA.

      On an average our PRP sample will have 100 billion platelets and 30x concentrate of blood
      platelet in 2ml of final PRP.

      Exosomes preparation:

      Sixty ml of blood in EDTA-coated tubes will be spin at 1,200 g for 10 minutes at 4°C to
      separate plasma. Plasma will be filtered with (0.22 μm) filter. Avoid any pelleted material
      while transferring the plasma. Discard the pelleted material. This tube has special filter to
      concentrate exosomes and other growth promoting proteins. Tube will be centrifuged at 3000 g,
      RT, 30 min and exosomes will be collected and then will be concentrated (0.5 ml to 3ml). To
      recover exosomes clogged in wall of filter -add 20 ml NS (preferably PBS) into micro
      centrifuge tube and rinsed it by centrifugation at 3,500 g. Collect the concentrated soup of
      exosomes again which were clogged in filter wall.

      Quantification of protein content will be done by bicinchoninic acid (BCA) assay.

      Nanoparticle tracking analysis (NTA) Vesicle suspensions with concentrations between 1×107/ml
      and 1×109/ml will be examined using a Nanosight NS300 (NanoSight Ltd., UK) equipped with a
      405 nm laser to determine the size and quantity of particles isolated. A video of 60-sec
      duration will be taken with a frame rate of 30 frames/sec, and particle movement will be
      analyzed using NTA software (version 2.3; NanoSight Ltd.).

      Western blot analysis The exosome supernatant will be denatured in sample buffer and will be
      subjected to western blot analysis. Purified exosomes will be analysed for protein expression
      HSP70, flottilin, CD63, TSG101 and calnexin. The proteins were visualized on the Bio-Rad
      ChemiDoc XRS Imager system (Bio-Rad Laboratories, Berkeley, CA, USA).

      Study Design Patients will be randomized into 2 arms with either to receive PRP with exosomes
      at the centre of the nucleus pulposus or equal amount of placebo and will be evaluated by 2
      interventional spine and medicine physicians.

      Randomisation and Arms of the study:

      Approximately Three hundred patients will be screened to randomly allocate to treatment or
      control groups at a 1:1 ratio, to assign 30 patients into each 4 arm:

        1. Arm I will be assigned to receive placebo (trigger point injection under C arm)

        2. Arm II: to receive PRP with exosomes at the center of the nucleus pulposus (2ml) Test
           product(s), dose and mode of administration duration of treatment Patients will be
           randomly selected based on computer generated number to receive one undistinguishable
           injection of PRP ex or placebo. Following skin disinfection of the back 2 ml from the
           total PRPex procured was injected as soon as possible by an fluoroscopy guided lateral
           approach. Patients received the placebo in a similar manner but not to disc .it will be
           in muscles zone around trigger point .Physician who evaluated the study and patient were
           unknown of treatment. Patients were advised to continue with usual exercises, routine
           precautions, physiotherapy and back care.

           Paracetamol (to a maximum of up to one gram) three times a day, will be allowed to be
           used as a rescue drug. No systemic corticosteroid, potential symptom-modifying drug
           /procedure will be allowed during the study.

           Study assessments / Outcome measures

             1. Visual analog scale (VAS)

             2. Roland Morris Disability Questionnaire (RDQ)

             3. SF 36 health questionnaire

             4. Functional rating index

             5. MRI - disc degeneration grading, T2 quantification any evidence of discitis
                Patients will be assessed by visual analog scale (VAS), SF 36 health questionnaire,
                Functional rating index at baseline and followed at 1, 3, 6, and 12 months. The
                structural efficacy will evaluated by MRI t2 quantification and grading as
                described earlier in literature at baseline and at 6 months by two experienced
                radiologists. Assessors will be blinded.

                Monitoring visits will be conducted according to standard monitoring procedures.

                Discontinuation from treatment with a specific reason will be ascertained. Any
                evidence of infection/discitis will be delicately observed. Worsening of pain will
                be deeply analyzed.

                The patients will be evaluated for any adverse effects and vital signs once a week
                for the first month and then once a month up to 6 months. Blood samples were
                collected at the time of entry into the study, at six months, and on a final visit
                to examine biological variables as well as liver and kidney functions. Tolerability
                was assessed and laboratory-based haematological and biochemical assays were
                performed. A possible causal relationship with the treatment arm will also be
                assessed.

                STATISTICAL ANALYSIS:

                Analysis of variance (ANOVA) will be used to investigate the differences between
                groups. Differences in paired samples will be compared using the two-tailed
                Student's t-test. Correlations between variables will be assessed by Pearson's
                correlation. SPSS 20.0 will be used for statistical analyses (SPSS, Inc., Chicago,
                IL, USA). P<0.05 will be considered to be statistically significant.

                Identification of the relationship between safety issues/side effects and arousal
                level using measurement by rating scales. To test whether the relationship between
                safety issues/side effects and arousal level differs between IBRF ACP/MCP vs.
                Standard of Care groups, using measurement by rating scales, two analyses will be
                conducted. Canonical correlation will be used to determine which side effect (or
                combination of side effects) explains variance in the rating scale measurements.
                Additionally, cluster analysis will be employed to determine whether side effects
                are similarly related to arousal levels and outcomes for patients in the three
                treatment groups.

                MONITORING PROCEDURES/ADVERSE EVENTS / SAFETY ENDPOINTS Monitoring The monitors
                will visit the center during the study in addition to maintaining frequent
                telephone and written communication.

                The following guidelines meet the requirements specified in 21 CFR §812.46 and are
                provided to describe the procedures for monitoring the clinical studies. If the
                investigator is not complying with the signed Investigator Agreement, the protocol,
                or any condition of the study (e.g., incomplete data forms), the monitor has the
                right to terminate the investigator's participation in the study.

                Pre-Study Monitoring Procedures

                The designate (i.e. monitor) will be responsible for determining and documenting
                that each investigator clearly understands and accepts the responsibilities and
                obligations incurred in conducting a clinical study. The monitor will ensure prior
                to study initiation that the investigator:

                • Understands the requirements for a well-controlled study;

                • Understands the nature of the clinical protocol

                • Understands his/her reporting obligations;

                • Understands the requirements for device accountability;

                • Understands and accepts the obligations to obtain informed consent in accordance
                with 21 CFR Parts 50 and 56;

                • Understands and accepts the obligation to obtain IRB review and approval of the
                clinical investigation before it is initiated and to ensure continuing review of
                the study by the IRB in accordance with 21 CFR Part 56, and to keep the sponsor
                informed of all IRB actions concerning the study;

                  -  Understands and accepts the requirements regarding financial disclosure of
                     clinical investigations, 21 CFR Part 54;

                  -  Has adequate facilities and access to an adequate number of suitable subjects
                     to conduct the investigation;

                  -  Has the required documentation on file, including IRB approval and a signed
                     investigator agreement.

                Periodic Monitoring Visits • Monitoring visits will be conducted in accordance with
                the FDA Guideline for the Monitoring of Clinical Investigations - January 1988.

                • The monitor should visit each site regularly to ensure the following:

                • Facilities continue to be adequate and acceptable.

                • The protocol is being properly followed.

                • The IRB/IEC has approved or been notified of any protocol changes.

                • Accurate, complete and current records are being maintained, and the information
                recorded.

                • Accurate, complete and timely adverse event reports are being submitted

                • Informed consent has been obtained.

                • The reason for a subject's withdrawal from the study has been documented.

                • Reports are being submitted to the IRB and sponsor.

                • The appropriate staff is carrying out study activities.

                • All case report forms and other documentation related to the study will be
                reviewed upon receipt, and the site will be promptly notified of any deficiencies.

                Frequency of Monitoring Visits a) Monitoring visits will be conducted according to
                standard monitoring procedures. Monitoring visits will be determined on the basis
                of several factors, including: b) Duration of the study; c) Number of outstanding
                issues from previous visits; d) Number of subjects enrolled; e) Number of
                investigators/sites; and, f) Complexity of the study.

                Study Termination All routine monitoring functions must be performed prior to the
                study termination visit. The study termination visit may be combined with a
                monitoring visit. The following tasks should be completed at the last visit by the
                monitor.

             1. Remind the investigator of the obligation to retain the records. b) Prepare final
                monitoring report for IEC.

           Reports of Monitoring Visits

           Monitoring reports must be completed for all visits. Reports must include the following
           information:

           a) Date of the visit; b) List of study site personnel present; c) A summary of the
           findings, problems
    

Intervention name(s): Platelet rich plasma (PRP) with exosomes; Normal Saline
Intervention type(s): Biological; Drug
Intervention description(s): Autologous Platelet rich plasma (PRP) with exosomes Following skin disinfection of the back 2 ml from the total PRPex procured will be injected as soon as possible by an fluoroscopy guided lateral approach.; placebo : 2 ml normal saline para discal administration
Allocation: Randomized
Intervention model: Parallel Assignment
Intervention model description: Arm I will be assigned to receive placebo (trigger point injection under carm)
Arm II: to receive PRP with exosomes at the center of the nucleus pulposus (2ml)
Masking: Triple (Participant, Investigator, Outcomes Assessor)
Primary purpose: Treatment
Keywords: intra-discal PRPEx Chronic Low Back Pain