Clinical trial NCT02387749: Effect Of Mesenchymal Stem Cells Transfusion on the Diabetic Peripheral Neuropathy Patients .
Official title: Effect Of Mesenchymal Stem Cells Transfusion on the Peripheral Neuropathy in Diabetic Patients Measured by Nerve Conduction.
Study type: INTERVENTIONAL
Overall status: COMPLETED
Phase: NA
Conditions: Diabetic Peripheral Neuropathy
Keywords: diabetic peripheral neuropathy
mesenchymal stem cells
Lead sponsor: Cairo University
Sponsor class: OTHER
First posted: 2015-03-13
Enrollment: 10
Sex: ALL
Minimum age (years): 18
Maximum age (years): 45
Standard ages: ADULT
Accepts healthy volunteers: no
Allocation: NA
Intervention model: SINGLE_GROUP
Primary purpose: TREATMENT

Brief summary: A debilitating consequence of diabetes mellitus (DM) is neuropathy which globally affects between 20 -30% of diabetic patients and up to 50% in other studies. The incidence of diabetic neuropathy (DN) is estimated to be up to 45% for type 2 diabetic patients and 59% for type 1diabetic patients in USA.(DN) is the most common complication of DM.The pathophysiology of DN is promoted by several risk factors: micro vascular disease, neural hypoxia, and hyperglycemia-induced effects.At the molecular level, the primary cause of diabetic complications is known to be hyperglycemia, which disrupts cellular metabolism by the formation of reactive oxygen species (ROS).In the aspect of nerve functions, ROS formation increases neuron's susceptibility to damage. In addition, hyperglycemia impedes production of angiogenic and neurotrophic growth factors, which are necessary for normal function of neurons and glial cells and maintenance of vascular structure.No definitive disease-modifying treatments have been to reverse DN. The current treatment focuses on tight glycemic control which can reduce potential risk factors for further nerve damage and DN-associated pain management.In many studies, deficiency of neurotrophic factors and lack of vascular support have been regarded as key factors in the development DN.Mesenchymal stem cells (MSCs) are particularly attractive therapeutic agents because of their ability to self-renew, differentiate into multi lineage cell types, and locally secrete angiogenic cytokines, including basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF) .These factors were reported to prompt neovascularization and have support for neural regeneration.It was plausible that MSCs may also be an effective therapeutic agent for the DN treatment through the paracrine effects of bFGF (Shibata et al., 2008) and VEGF and their potential to differentiate into neural cells such as astrocytes, oligodendrocytes , and Schwann cells.The adherent nature of MSCs makes them easy to expand in culture and an attractive candidate to use in cell therapy.Therefore, cell therapy has recently emerged as an attractive therapeutic strategy to meet the needs of both neurotrophic and vascular deficiencies of DN.Proper diagnosis of DN depends on the pattern of sensory loss, reflex test, electrodiagnostic studies, and imaging

Detailed description: Objectives This study will be conducted to investigate the effects of Mesenchymal stem cells (MSC) transfusion on diabetic peripheral neuropathy in diabetic patient. (MSCs) have been reported to secrete various cytokines that exhibit angiogenic and neuro supportive effects.

Study Design Experimental interventional study. phase II clinical trial

Ethical committee approval (was it ethically approved by the department) Internal medicine department Yes

Study Methods

Population of study \& disease condition (e.g women with hepatitis, .........) Diabetic patients(type I, type II) with documented peripheral neuropathy as determined by impaired nerve conduction

Inclusion criteria:

(Type I, type II) diabetic patients age range (18-45) years, with diabetic peripheral neuropathy proved by clinical assessment and nerve conduction who did not receive treatment for diabetic peripheral neuropathy.

Exclusion criteria:

Decompensated cardiac, renal or liver disease. Associated autoimmune diseases Associated endocrinal diseases Pregnancy, usage of contraceptive pills or steroids.

Methodology in details The study will be conducted on patients with diabetic peripheral neuropathy collected from internal medicine department(inpatient and out patients, males and females)

All subjects of this study will be submitted to the following :( preparatory visit before (MSCs) transfusion visit.)

* Thorough clinical assessment as a general assessment of the patient, also assessment of diabetes complications especially diabetic neuropathy. This is including full history and examinations (e.g., pain, sensory loss, ulcers, sensory level, etc....) this preparatory assessment will be done at internal medicine department.
* Venous blood will be drawn in the morning after an overnight fast in the pre (MSCs) transfusion visit.

Plasma biochemical blood measurements will be determined by standard laboratory procedures in the central lab at clinical pathology department, Kasr Alaini hospital)

* Fasting blood glucose level, 2 hours postprandial.
* C-peptide.
* Hb A1C.
* Basic Fibroblast Growth Factor (bFGF), vascular endothelial growth factor (VEGF) by ELISA.
* Complete blood picture.
* Liver functions in the form of ALT, AST
* Kidney functions in the form of serum creatinine.

  * fundus examination
  * Nerve conduction study at neurophysiology unit.
  * Bone marrow aspiration of about 90 ml under local anesthesia once at the first visit after preparatory visit mentioned before, the patient will be admitted for 12 hours and will be monitored closely to avoid anaphylaxis (by steroids, anti-allergic treatment), if no complication, will be discharged.

To avoid infection: During bone marrow aspiration, procedure will be done under complete aseptic precautions, placed in sterile tubes containing pre-servative-free heparin (Sigma-Aldrich, St. Louis, USA) Separation and processing of the sample will be done under good manufacture procedure (GMP): Bone Marrow Aspirate (BMA) will be withdrawn under good sterilization of the skin in an isolated area. Processing of the sample will be done in the laminar air flow; all supplies are disposable and sterile.

Technique:

Separation of mononuclear cells:

The bone marrow aspirate will be diluted at a ratio of 6:1 with phosphate buffer saline (PBS) with 2 mM EDTA (30 ml BM aspirate+ 5 ml PBS/EDTA buffer). The MNCs will be separated under aseptic conditions using a Ficoll. Hypaque desity gradient (density 1.077, GibcoBRL, Grand Islan, NY, USA) by centrifugation at 1800 rpm for 20 min then the MNCs will be plated in 40 ml alpha-modi-field Eagle's medium (αMEM), serum free media; mesencult(Mesenchymal stem cell culture),penicillin (100 U/ml),streptomycin(10 mg/ml),0.5 ml amphotericin B(all from Gibco BRL) and 10 ng/ml basic fibroblast growth factor (b-FGF) (R\&D system, Minneapolis, MN) and will be incubated at 370 c in a humidified atmosphere containing 5% CO2 (Digirolamo et al.1999).after one day ,non adherent cells will be cultured in the presence of Mesenchymal media for 3 weeks changed every 1 week (cambrex Bioscience ,Nottingham, uk). After reaching 80% confluence the MSCs will be placed in 10 ml saline and will be infused intravenously

Flow cytometry Surface expression of MSCs using anti- (CD271, CD34, CD73, CD90, CD105, CD29) monoclonal antibodies (mAbs) will be analyzed using flow cytometry. MSCs (2X105 cells) will be suspended in PBS containing 1% BSA and will be stained with flurochrome -conjugated mAbs for 20 minutes on ice (anti-mouse mAanti-CD 271, CD34 CD73, CD90, CD105,CD29; BD Bioscience, MN, USA).flow cytometric analysis will be performed using a FACSCaliber (BD bioscience)equipped with cell Quest software.10000 cells will be passed in front of the laser for each sample. Each sample will be analyzed in duplicate. A cut off value at 20% will be set to categorize samples as positive.

Mesenchymal stem cells will be identified by morphology and immunophenotyping in the central lab at clinical pathology department, Kasr Alaini hospital( stem cell lab).

Mesenchymal stem cells transfusion slowly intravenous will be applied after these procedures for the patients after taking their approval and informed consent.

Follow up 3 months after Mesenchymal stem cells transfusion by fasting blood glucose level, 2 hours postprandial, C-peptide, Hb A1C, (bFGF), (VEGF) and nerve conduction at kasr Alaini hospital departments as mentioned before.

Possible Risk (mention if there is any risk or not) Anaphylaxis Infection

Primary outcomes (Most important outcomes to be assessed)

1- Effect of mesenchymal stem cells transfusion on diabetic peripheral neuropathy regarding improvement of clinical symptoms like pain, sensory loss and improvement of nerve conduction.

Secondary outcome parameters (other outcomes to be assessed)

1. Mesenchymal stem cells being a treatment modality in diabetes complications like peripheral neuropathy, to avoid hazards on patients secondary to diabetic peripheral neuropathy.
2. Improving of diabetic condition like lowering of blood glucose level, decrease in insulin requirements and improvement of general condition.

Sample size (number of participants included) 10 diabetic patients with diabetic peripheral neuropathy .

Source of funding (is there any source of funds or not) Faculty Of Medicine, Cairo University.

Time plan (when to start/ when expected to finish/ when to publish) At least 20 months

Eligibility criteria: Inclusion Criteria:

* (Type I, type II) diabetic patients age range (18-45) years, with diabetic peripheral neuropathy proved by clinical assessment and nerve conduction who did not receive treatment for diabetic peripheral neuropathy.

Exclusion Criteria:

* Decompensated cardiac, renal or liver disease. Associated autoimmune diseases Associated endocrinal diseases Pregnancy, usage of contraceptive pills or steroids.

Primary outcomes:
measure: Measurement of b-FGF, v-EGF MEASURED BY ELISA
timeFrame: zero ( before) , 7 DAYS, 90 days
measure: Change of Nerve Conduction Velocities of Nerves Affected Measured by Nerve Conduction Study.
timeFrame: base line(zero dya), 90 days after stem cells transfusion.
measure: Change of Nerve Conduction Latency of Nerves Affected Measured by Nerve Conduction Study
timeFrame: base line(zero dya), 90 days after stem cells transfusion .
measure: Change of Nerve Conduction Amplitude of Nerves Affected Measured by Nerve Conduction Study.
timeFrame: base line(zero dya), 90 days after stem cells transfusion
Secondary outcomes:
measure: Change of Levels of Fasting Blood Sugar and 2 Hours Post Prandial at Base Line ( Zero Day ) and After (90 Days) After Stem Cells Transfusion
timeFrame: base line (zero day) and 90 days after stem cells transfusion
measure: Change of Levels of Glycated Haemoglobin( HA1C) After Stem Cells Transfusion Measured in Percent %
timeFrame: at base line (zero day) and 90 days after stem cells transfusion
Other outcomes:
Locations:
Study officials:
affiliation: Cairo University
name: Mohamed Gamal ElDin Saadi, phd
role: STUDY_CHAIR
affiliation: Cairo University
name: Dina Abdelmagid, MD, MRCP UK
role: PRINCIPAL_INVESTIGATOR
References:
citation: Jackson L, Jones DR, Scotting P, Sottile V. Adult mesenchymal stem cells: differentiation potential and therapeutic applications. J Postgrad Med. 2007 Apr-Jun;53(2):121-7. doi: 10.4103/0022-3859.32215.
citation: Keilhoff G, Stang F, Goihl A, Wolf G, Fansa H. Transdifferentiated mesenchymal stem cells as alternative therapy in supporting nerve regeneration and myelination. Cell Mol Neurobiol. 2006 Oct-Nov;26(7-8):1235-52. doi: 10.1007/s10571-006-9029-9. Epub 2006 Jun 16.
citation: Kinnaird T, Stabile E, Burnett MS, Epstein SE. Bone-marrow-derived cells for enhancing collateral development: mechanisms, animal data, and initial clinical experiences. Circ Res. 2004 Aug 20;95(4):354-63. doi: 10.1161/01.RES.0000137878.26174.66.
citation: Morbach S, Lutale JK, Viswanathan V, Mollenberg J, Ochs HR, Rajashekar S, Ramachandran A, Abbas ZG. Regional differences in risk factors and clinical presentation of diabetic foot lesions. Diabet Med. 2004 Jan;21(1):91-5. doi: 10.1046/j.1464-5491.2003.01069.x.
citation: Nakae M, Kamiya H, Naruse K, Horio N, Ito Y, Mizubayashi R, Hamada Y, Nakashima E, Akiyama N, Kobayashi Y, Watarai A, Kimura N, Horiguchi M, Tabata Y, Oiso Y, Nakamura J. Effects of basic fibroblast growth factor on experimental diabetic neuropathy in rats. Diabetes. 2006 May;55(5):1470-7. doi: 10.2337/db05-1160.
citation: Rathur HM, Boulton AJ. Recent advances in the diagnosis and management of diabetic neuropathy. J Bone Joint Surg Br. 2005 Dec;87(12):1605-10. doi: 10.1302/0301-620X.87B12.16710. No abstract available.
citation: Shibata T, Naruse K, Kamiya H, Kozakae M, Kondo M, Yasuda Y, Nakamura N, Ota K, Tosaki T, Matsuki T, Nakashima E, Hamada Y, Oiso Y, Nakamura J. Transplantation of bone marrow-derived mesenchymal stem cells improves diabetic polyneuropathy in rats. Diabetes. 2008 Nov;57(11):3099-107. doi: 10.2337/db08-0031. Epub 2008 Aug 26.
citation: Adler AI, Stratton IM, Neil HA, Yudkin JS, Matthews DR, Cull CA, Wright AD, Turner RC, Holman RR. Association of systolic blood pressure with macrovascular and microvascular complications of type 2 diabetes (UKPDS 36): prospective observational study. BMJ. 2000 Aug 12;321(7258):412-9. doi: 10.1136/bmj.321.7258.412.
citation: Digirolamo CM, Stokes D, Colter D, Phinney DG, Class R, Prockop DJ. Propagation and senescence of human marrow stromal cells in culture: a simple colony-forming assay identifies samples with the greatest potential to propagate and differentiate. Br J Haematol. 1999 Nov;107(2):275-81. doi: 10.1046/j.1365-2141.1999.01715.x.
citation: Dalla Paola L, Faglia E. Treatment of diabetic foot ulcer: an overview strategies for clinical approach. Curr Diabetes Rev. 2006 Nov;2(4):431-47. doi: 10.2174/1573399810602040431.