The Stowe Foundation

Cardiac Care Trial Phase II

A PHASE TWO CLINICAL TRIAL OF THE SAFETY AND FEASIBILITY OF BONE MARRROW STEM CELLS AND THE DEVELOPMENT OF CARDIOVASCULAR REGENERATION IN PATIENTS WITH MYOCARDIAL INFARCTION.

Ischemic cardiopathy is one of the most frequent causes of death in the world as well as being a severe public health problem. A heart that suffers a myocardial infarction is at great risk because the heart muscle does not have the capacity to form new tissue, this results in an area of necrosis and leads to fibrous scarring.

As a consequence of this scarring and modified ventricular function the infarction may lead to a condition known as congestive heart failure  (CHF).

Myocardial infarction is now considered one of the gravest public health problems that exist today due to its frequency, its social and economic impact and most of all because of the adverse effect it has on the patients quality of life.

Myocardial Infarction ( M. I .)occurs in one percent of the general public who live in the United States and England and between 5 and ten percent of those over the age of 75.  The National Institute of Statistics Geography and Informatic ( Instituto  Nacional de Estadística, Geografía e Informática, INEGI) estimates that in México myocardial infarction is responsible for 4.3 percent of all hospitalized patients over the age of 45 , and 75 percent in those patients over the age of 65.  Re-cent data from the Framingham Study indicates that mortality after 5 years of having suffereed an MI was 75 percent in men and 65 percent in women, compared to an average of 50 percent in all types of cancer, (Navarro- López et al, 1999).

The past few years have brought with them new procedures to treat the acute phase of an M .I., such as fibrinolysis and primary angioplasty and these have had a significant impact on the prognosis of these patients.  However, a high percentage of these patients develop cardiac insufficiency.  Important advances have also been made in the treatment of these patients with beta blockers, IECAS and espirono lactona which retard the onset of cardiac insufficiency as well as surgical procedures such as dynamic cardiomyoplasty and mechanical assistance. Nevertheless, while these new procedures do retard the advance of cardiac insufficiency, it still progresses to a terminal phase. The only real resource left to treat these patients is to resort to cardiac transplantation  of which are important limitations such as the law of supply and demand as well as the need for patients  use inmune-suppressors the rest of their lives.

The possibility of inducing the development of cardiomyocytes in an adult heart has been considered a promising strategy in the treatment of cardiac insufficiency, ventricular hypertrophy or ischemic cardiac disease (Taylor and cols, 2000) . This procedure has been deemed inappropiate however, because cardiac cells have specific characteristics which incapacitate them from entering into a cellular cycle of division and reproduction. Cardiomyocytes are created from a cellular precursor which divides and forms cells of its own type.  During fetal life, these cells differentiate and contractile myofibers appear in their cytoplasm.  These fetal contractile cardiomyocites retain the capacity to divide in spite of being in a differentiated state.  In the case of human beings, this capacity is retained up until three to four months post partum ( Kessler and cols,  1999). Generally speaking, we know that for a few months after birth we possess the maximum number of cardiomyocytes that we will ever have and that from this moment on the loss of these cells will not replaced.  This progressive decrease in the number of these cells eventuallly results in death.  This concpet of the heart as a non -regenerative organ is based on a superficial observations and is contrary  to recently obtained data from experiments conducted on both animlas and humans that show that the heart is a regenerative organ which constantly augments the production of new cells in response to differenmt physiological stimuli , (Kessler and clos, 2001).  Nadal..Ginard and colleagues demostrated in 2000 that over a four to six month periof, approximately one third of cardiac cells are replaced, this signifies that in two to three years the entire organ would be renewed. In all probability, a 50 to 60 year old person may have a heart in which the majority of the cardiomyocytess are no older that four or five years. This would indicate that the heart does seem to have the ability to rejuvenate.

 

When a person suffers an M. I. , the rapid opening of the occluded artery has significantly reduced the number of sudden death sand diminished the clinical secuelae, (Herreros and cols, 1994). Nevertheless, in spite of the rapid reestablishment of perfusion to the heart, post infarct cardiac insufficiency continues to be a major problem. This malady results from the process of ventricular remodeling characterized by progressive expansion of the occluded area and dilation of the left auricular cavity, ( Dorfran and cols,  1998 ). The principal objective of reverting the ventricular remodeling would be an incrcease in the regeneration of cardiomyocytes as well as revascularization within  the occluded area.

 

Research has reported the characterization and isolation of a special kind of cell,  known as Embryonic Stem Cells ( ESC). These cells have the capacity to renew themselves and to remain in a multi_potent condition which means that they have the capacity, in vivo, to originate more than 200 specialized types of cells derived from three embryonic germinal lines: ectoderm, mesoderm and endoderm.  Because of this, the multi-faceted capacity of the ESC gives rise to the expectation of developing cellular transplants to treat many pathologies among which are myocardial infarctions, ( Donovan and cols,2001) .

 

Embryonic stem cells have been obtained in vitro from the Inner Cell Mass (INC), of mammal blastocytes sucha as rats, primates and humans. They are cultivated over singular layers of MYTOTIC inactived mouse embryonic fibroblasts, MEF, which provide factors, up till undentified, which maintain the ESC in a permanently undefined state, ( Thomson and cols, 1995, 1998 and Odorico and cols , 2001) . However, when ESC are cultivated without the single layer of MEF cells they form multi-cell groups called Embryonic Bodies or EB, which are a mixture of cells with diverse phenotypes such as neuron, cardiac, muscle, etc..  This demonstrates that the spontaneuos differentiation of ESC occurs in a totally disorganized manner given that the EB are made up of a cellular mixture which contains neuron, cardiac, etc, cells.

 

On a purely experimental level, diverse research groups have developed strategies to induce the differentiation of ESC toward a determined type of specialized cells. Two approaches have been used :

 

  1. By the use of factors which induce or regulate determined programs of differentiation, such as growth factor of neurons and the growth factor of fibroblasts, NGF and FGF respectively , which are used as program inductors in neuron differentiation, (Lovell-Badge and cols, 2001 and Odorico and cols,  2001).
  2. Through molecular biology procedures such as the incorporation of an active gene into the genome of the ESC, which actives a state of specific differentiation.  This procedure is used however, only when the gene identified defines the differentiation of interest, ( Kischstein and cols, 2001).

 

Through the use of these procedures, differentiation of the ESC of human and rats to diverse specialized cells with the objetive of designing cellular treatment for various pathologies has been accomplished. It has been possible to obtain vascular cells (Yamashita and cols,2000), neurons  which produce dopamine and serotonin for the treatment  of Parkinson´s disease (Lee and cols 2000)neurons for the treatment of spinal lesions (Ogawa and cols, 2002), Espinosa-Jeffrey and cols, 2002, Wichterie and cols , 2002, Teng and cols, 2002) insulin cells for the treatment of diabetes (Lumelsky and cols, 2002, Zulewsky and cols, 2001, Assady and cols, 2001) and of special interest to us cardiocytes with contractile activity for the treatment of myocardial infarction (Doetschman and cols, 1993, Maltsev and cols, 1999, Reinecke and cols, 1999  and Mullerand cols, 2002) . However, even with all the advances in cellular replacement therapy that have been made, the ethical question must be taken into consideration.The isolation of ESC requires the disintegration of frozen human embryos. This has forced distinct research groups to direct their efforts to find and identify new cells with the same multi-potential capacity from a different origin than the ESC.

NEW  SOURCES

HEMATOPOIETIC  PROGENITOR  CELLS.

 

In recent years, a new kind of cell has been identified which we now know as a common or shared precursory cell referred to as Hematopoietic multipotential stem cell or Hematopoietic Progenitor Cells, HPC, also known as Stem Cells located in the bone marrow. Beginning with this common precursor, Lymphoid stem cells and pluri-potential myeloid stem cells produce lymphocytes and stem cells respectively. The LSC seem to be the origin of the  precursors of B and T cells, (pre –B and pre-T). From the pluri-potential MSC four types of unipotential stem cells originate   by specific factor stimulation capable of causing the differentiation into  erythroides, megacardiocites, eosinophylic and granulocytic-macrophagic, also know as Colony Forming Units.

 

From the variety of involved stem cells come the precursors, recognizedby their morphology, along the differential cellular lines such as pro-erythroblasts, myeloblasts or mega-cardioblasts . Because mature blood elements have a limited life span, they need to be constantly replaced which is made possible by the stem cells ability to reproduce. Pluri-potential stem cells have the maximum potential for auto-renovation, however, the majority of these cells are found to be in past, their active phase of cellular activity.  While they commit to a differential line their renovation capacity is limited but even at that, a

large portion of active stem cells are found.

 

Though the morphology of the HPC is unknown, research has establised the presence of an antigenic monoclonal marker of the cellular surface which is known as CD34.  This is a trans-membranal protein of the cellular surface that forms the HPC and the endothelia vascular cells of some tissues and their presence on the cellular surface is in inverse relation to the state of differentiation since their presence is lost in the compromised progenitor phase and the functional signific ance of its presence is unknown. It is expressed in one to five percent of bone marrow cells in the adult human, in one percent of the mononuclear fraction of circulating blood cells and in two to ten percent of fetal bone marrow and  liver cells and aprroximately 90 to 95 percent of CD34 cells  present antigens indicative of a compromise towards lymphoid or myeloid series.

 

As opposed to ESC isolated from human embryos,  stem cells can be isolated directly from the patient´s bone marrow.The potential use of these cells in regenerative therapy and myocardial infarction has been demonstrated with the administration of bone marrow mononuclear BMC cells with intracardiac injection and in experiments with rats whose coronary arteries have been manually occluded.  Detailed analysis revealed that the BMC  can differentiate into endothelial cells thus increasing vascularization, promoting the repair of damaged tissue by generating cardiomiocytes, producing citokines and preventing the development of fibrosis, (Orlic and cols, 2001 and 2002).  Seven  Phase 1  published studies in humans have shown that autologous BMC   transplants injected directly into the area of damaged myocardial tissue or infused  on the edge of the same have shown a marked improvement in vascularization and cardiac perfusion, thus improving function of the damaged heart. These results were documented by studying the patient´s quality of life, use of medication and Magnetic Resonancy Imaging, MRI( TSF and cols, and Stam and cols, 2003). These studies provide a rational for further clinical research .

 

Based on the above, this project will develop and establish a protocol for the point of care isolation of stem cells from bone marrow aspiration. These cells will be used in a Phase II Clinical Trial to  evaluate the safety and clinical potenntial of transplanting BMC`s as a treatment to induce cardiovasculogenesis in patients with myocardial infarction.

 

OBJECTIVES

GENERAL OBJECTIVES

Isolation and characterization of bone marrow stem cells

 

Phase II Clinical Trial to determine the safety and feasibility of transplanting bone marrow stem cells for the regeneration of ischemic heart muscle to improve heart function in patients who have suffered myocardial infarctions.

 

SPECIFIC  OBJECTIVES

 

  •  Reduction of infarct size
  • Increase in global left ventricular ejection fraction
  • Increase in infarction wall velocity
  • Improvement in maximum oxygen uptake
  • Improvement in regional F fluor dexosy glucose uptake into infarct tissue.

 

SCIENTIFIC PROTOCOL AND METHODOLOGY OF THE SAFETY AND  FEASIBILITY PHASE OF BONE MARROW STEM CELL TRANSPLANT  AFTER  MYOCARDIAL INFARCTION.

 

MOLECULAR   CHARACTERIZATION  OF  B.M.C.

 

It is very important to prove the correct identity of a cell line when processing its characterizations. This will be done by identifying protein molecular markers  which present themselves only in the type of cell that is to be cultivated. The molecular markers used for bone marrow cells will mainly be a CD34positive cells, AC 133positive cells and CD45/CD14 negative cells. Receptor. The presence of the marker will ve evaluated by detecting protein products using a flow Cytometer and specific antibodies to each protein.

 

PROTOCOL FOR ASPIRATING BONE MARROW FOR CONCENTRATION USING THE HARVEST TECHNOLOGY CONCENTRATION SYSTEM

The concentration process involves three separate but related procedures: (1) the aspiration procedure, (2) the filtration procedure and (3) the concentration procedure.  The following protocol describes the specific activities required in procedure.

 

ASPIRATION PROCEDURE:  (Conducted within the sterile field)

 

Conducted by a Hematologist and assisted by Hematology Tech or assisting nurse

 

FILTRATION PROCEDURE  (Conducted within the sterile field)

 

Conducted by Hematology Tech or assisting nurse

 

CONCENTRATION PROCEDURE  (Conducted outside the sterile field)

 

Conducted by Circulating Nurse

 

NOTE: If the processed bone marrow aspirate is intended for infusion, patients MUST be on an antiplatelet drug regiment 72 hours prior to infusion.

 

PREPARATION OF ASPIRATION MATERIALS:  (Performed within the sterile field)

 

  • Activities performed by Hematology Tech or assisting nurse

 

Materials:

 

  • Two aspiration needles with removable stylets
  • Two replacement stylets
  • ACD anticoagulant solution and stainless steel bowl to rinse aspiration needles and stylets – NOT IN HARVEST TECHNOLOGIES KIT
  • 10 ml syringe for rinsing aspiration needles with ACD solution – NOT IN HARVEST TECHNOLOGIES KIT
  • Eight 20 ml syringes with needles attached
  • Five ml of heparin in concentration of 1,000 units / ml – NOT IN HARVEST TECHNOLOGIES KIT
  • Sterile sample cup for heparin
  • Harvest Technology filter assembly (sterile)
    1. 260 micron filter
    2. 60 ml collection syringe
    3. blunt needle for collection syringe 

 

PREPARATION OF ASPIRATION MATERIALS:  (Performed within the sterile field)

 

Activities performed by Hematology Tech or assisting nurse

 

 

Activities:  (Performed within the sterile field)

 

 

  • Prepare eight 20 ml aspiration syringes

 

  1. Coat each 20 ml syringe with heparin as follows:
    1. Draw 0.5 ml of the 1,000 units per ml heparin solution into the syringe
    2. Pull the plunger back to its fully extended position
  • Invert the syringe several times to insure that heparin has coated the entire inner surface of the syringe
  1. Push the plunger back in to remove any air and remove the attached needle

 

  • Prepare filter and collection syringe assembly

 

  1. Draw 1.0 ml of 1,000 units of heparin into the 60 ml collection syringe. Pull the plunger back to its fully extended position. Invert the syringe several times to insure the heparin has coated the entire inner surface of the collection syringe.

 

  1. Attach filter to 60 ml collection syringe. (NOTE: filter element should be proximal to the collection syringe for proper flow from aspiration syringe to collection syringe)

 

  1. Express the heparin from the syringe through the filter assembly. Invert the assembly several times to insure the heparin has coated the entire inner surface of the filter.

 

  1. Recover heparin back into the 60 ml collection syringe and remove the filter from the collection syringe. Express the remaining heparin into the clear sterile cup in the sterile field

 

  • Prepare aspiration needles

 

  1. Remove stylet from the aspiration needles.

 

  1. Aspirate ACD solution into 10 ml syringe. Flush ACD into the aspiration needle until it come out of the holes at the end of the needle. Coat the outside of the needle by placing it in the stainless steel basin with the ACD solution.

 

  1. Repeat steps (a) and (b) for second aspiration needle and each time after the needle has been used to obtain an aspirate.

 

  1. Place stylets in stainless steel basin with ACD solution

 

  1. Replace the stylet in the aspiration needles.

 

  1. The replacement stylets should not be placed in the ACD solution. These stylets should be used for aspirations on the opposite side.

 

ASPIRATION PROCEDURE:  (Performed within the sterile field)

 

  • Activities performed by Hematologist

 

Materials:

 

  • Two standard aspiration needles rinsed and coated with ACD solution
  • Two extra stylets to be used for aspirations from the right side of the ilium.
  • Eight 20 ml syringes prepared containing 0.5 ml of heparin at concentration of 1000 units / ml
  • Ten sterile syringe caps

 

ASPIRATION PROCEDURE:  (Performed within the sterile field)

 

  • Activities performed by Hematologist

 

Activities: (Performed within the sterile field)

 

  • Place patient in prone position on the procedure table.
  • Create visual access to the iliac crest by making a 1 – 2 cm stab incision over the left iliac crest.
  • Penetrate bone marrow of ilium using standard bone marrow aspiration needle with needle length of approximately 4 cm. Follow standard protocol for taking a bone marrow biopsy.
  • Upon penetration approximately 1 cm into the marrow cortex, remove the stylet from the needle and attach 20 ml syringe with heparin. The stylet should be placed in the ACD solution
  • Advance the aspiration needle / syringe assembly forward gently through the marrow until the needle contacts the cortical bone surface.
  • Aspirate 4 ml of bone marrow into the syringe
  • Pull the needle / syringe assembly 1 to 1.5 cm back into the marrow cortex and aspirate an additional 4 ml of bone marrow.
  • Remove syringe, pull back the syringe plunger back to allow for a 2 ml air bubble and invert the syringe six times to insure adequate mixing of the aspirate with the heparin and hand to Hematology Tech or assisting nurse.
  • Remove the aspiration syringe, flush the needle with ACD solution using the 10 ml syringe, remove the stylet from the ACD solution and replace the stylet into the needle.
  • Reposition the needle with the stylet and repeat steps 2 – 8.
  • Use two needles and stylets for four aspiration penetrations of the left ilium. Use replacement stylets and the two aspiration needles for the four aspiration penetrations of the right ilium
  • Procedure is complete after 60 ml of bone marrow has been aspirated from four penetrations of the left ilium and four penetrations of the right ilium
  • Activities performed by Hematology Tech or nurse assisting Hematologist in aspiration

 

Activities: (Performed within the sterile field)

 

After each aspiration the 20 ml aspiration syringes must be handled so as to prevent the formation of any clots.

 

  • Receive filled aspiration syringes from Hematologist, pull syringe plunger back to create a 2 ml air bubble and cap with sterile cap.
  • Gently invert the syringe six to eight times to insure through mixing of aspirate with the heparin.
  • Prior to beginning filtration process invert each syringe several times to insure proper mixing of heparin with the aspirate.

 

FILTRATION PROCEDURE:  (Performed within the sterile field)

 

  • Activities performed by Hematology Tech or nurse assisting Hematologist.

 

Materials:

 

  • 3 ml syringe and clear cup with heparin
  • 60 ml collection syringe with filter attached
  • Eight 20 ml syringes containing heparinized bone marrow aspirate
  • Blunt needle for 60 ml collection syringe

 

FILTRATION PROCEDURE:  (Performed within the sterile field)

 

  • Activities performed by Hematology Tech or nurse assisting Hematologist.

 

Activities:  (Performed within the sterile field)

 

  • Aspirate 0.5 ml of heparin from the clear sterile cup and express into the top of the filter assembly so that the heparin pools at the distal end of the filter assembly.
  • Attach a 20 ml aspiration syringe to the filter assembly
  • Filter aspirate by pushing aspiration syringe barrel and, when entire volume has been infused into the filter, pull gently on the barrel of the collection syringe to pull hold-up volume from the filter.
  • Repeat step 3 until total aspirated volume of 60 ml has been filtered into the collection syringe.
  • Remove collection syringe, attach blunt needle and pass syringe from sterile field to Circulating Nurse for concentration process.

 

CONCENTRATION PROCEDURE  (Performed outside the sterile field)

 

Performed by Circulating Nurse

 

Materials:

 

  • Harvest centrifuge
  • Processing kit (sterile)
  1. 30 ml bottle of ACD
  2. Two chambered processing disposable (PD)
  3. Three syringes
    1. 10 ml syringe with needle for transferring ACD into the PD
    2. 30 ml syringe with blunt cannula and BLUE spacer for removing excess plasma
  • 20 ml syringe with blunt cannula for removing concentrate
  • 5 ml syringe – NOT SUPPLIED IN HARVEST TECHNOLOGIES KIT
  • 5 ml of 4% sodium bicarbonate - NOT SUPPLIED IN HARVEST TECHNOLOGIES KIT
  • Sterile syringe caps
  • Blunt needle for 20 ml syringe containing BMC

  

CONCENTRATION PROCEDURE  (Performed outside the sterile field)

 

Performed by Circulating Nurse

 

ACTIVITIES:  (Performed outside the sterile field)

  • Peel back cover on sterile processing kit to expose PD and needles
  • Using a sterile alcohol or other antiseptic material wipe the top of the ACD bottle and the WHITE injection port of the PD.
  • Withdraw 4 ml of ACD into 10 ml syringe and inject into the plasma chamber of the PD (WHITE injection port of the PD)
  • Full collection syringe is transferred out of the sterile field with transfer blunt needle attached.
  • Using a sterile alcohol or other antiseptic material wipe the top of the RED injection port of the PD.
  • Inject the contents of the collection syringe into the marrow chamber of the PD. (RED injection port)
  • Open the lid of the centrifuge and put counterbalance in holder.
  • Place PD in holder. (NOTE: Be certain that the PD and counterbalance are properly seated within their holders)
  • Close lid and push green button to start process
  • Upon completion of processing cycle push LID button and open centrifuge lid
  • Remove PD and set back in holder within kit tray.
  • Using sterile alcohol or other antiseptic material, wipe the top of the WHITE injection port of PD.
  • Using the 30 ml syringe with the blunt cannula and blue spacer, remove excess plasma by aspirating until no further volume can be aspirated. The remaining volume in the chamber is 18 ml.  Remove the cannula and place a sterile cap on the 30 ml syringe.
  • Using sterile alcohol or other antiseptic material wipe the top of the WHITE injection port of PD. Prepare 5 ml syringe with 1.5 ml of 4% sodium bicarbonate and inject contents into the plasma chamber. This will result in a volume of 19.5 ml within the chamber.
  • Using the 20 ml syringe with the blunt cannula remove the remaining 19.5 ml volume from the chamber using the following procedure:
    1. First aspirate 3 to 5 ml into the syringe and gently express it back into the chamber in order to resuspend the cellular components back into the plasma.
    2. Repeat this action 4 or 5 times until the volume has a uniform consistence and then aspirate the entire volume into the syringe and withdraw the cannula from the chamber.
  • Remove the cannula and attach a sterile syringe cap and label syringe “BMC”.
  • Attach a blunt needle to the syringe to transfer the BMC back into the sterile field for infusion. The red sterile cup in the sterile field can be used to receive the BMC.

 

In Vitro Parameters for Testing Bone Marrow Aspirate Pre Processing and Post Processing after Bicarbonate Addition

 

  1. COMPLETE CELL COUNTS AND BONE MARROW MORPHOLOGY

An aliquot (0.5 ml recommended) of bone marrow aspirate pre processing and bone marrow concentrate post processing after bicarbonate addition are counted on an automated counter for Total Cell count. Supply copy of printout from hematology analyser

 

Smears are made from the samples and stained with Wrights-Giemsa stain.  A complete differential of bone marrow morphology should be reported.

 

  1. FLOW CYTOMETRY.

CD34 positive and CD133 positive and CD045/CD14 negative,  antigen expressing cells are measured on aliquots of bone marrow aspirate pre processing and post processing after bicarbonate addition viability staining.

 

  1. pH

Using a blood gas analyzer the pH of the bone marrow post processing after bicarbonate addition is measured.

 

  1. FIBROBLAST-COLONY FORMING UNITS (CFU-U)

CFU’s will be done on bone marrow aspirate pre and post processing after bicarbonate addition. Protocol is attached.

 

  1. GROWTH FACTOR ANALYSIS

PDGF-AB and TGF-b1, VEGF, and EGF.  Quantikine ELISA kits R&D Systems.

  1. a) Bovine Thrombin is prepared by injecting 5.0ml of 10% calcium chloride into a 5000U vial of freeze-dried thrombin. This will give you 1000U/ml for use.

 

  1. b) 0.5ml of processed bone marrow is placed into a glass tube. This is reacted with Bovine Thrombin (1000U/ml) in a 10:1 ratio (0.5ml BM plus 0.05ml thrombin) and incubated for 2 hour to release the growth factors.

 

  1. c) Centrifuge at 1700g for 8 minutes. Collect the supernatant which contains the growth factors. Freeze until time of analysis.

 

HUMAN BONE MARROW

 

Total Vol Bone Marrow Aspirate Processed:  ______

 

Total Vol Bone Marrow Concentrate: ______

 

Donor Marrow # _____

 

Pre Concentrate                                Post Concentrate

                                                           

 

            Total Number            %                       Total Number         %

 

Myeloblast

______________________________________________________

Promyelocyte

______________________________________________________

Myelocyte

_______________________________________________________

Metamyelocyte

_______________________________________________________

Band

_______________________________________________________

Segmented

_______________________________________________________

Neutrophil

_______________________________________________________

Eosinophil

________________________________________________________

Basophil

________________________________________________________

Lymphocyte

________________________________________________________

Monocyte

________________________________________________________

Plasma cell

________________________________________________________

Proerythroblast

________________________________________________________

Erythroblasts

________________________________________________________

Megakaryocyte

________________________________________________________

BSR

BioSciences Research Associates, Inc

 

STANDARD OPERATING PROCEDURES

 

DEPARTMENT:          Quality Systems

 

TITLE:                                  

                        

AUTHOR:      Robert Mandle                                              DATE:

TITLE:           Laboratory Director

APPROVAL:                                                                        DATE: 

TITLE:     Human Bone Marrow CFU-F

 

1.0 PURPOSE:        To provide a procedure for human bone marrow fibroblast colony forming units (CFU-F) as an indicator of stromal cell activity.

 

2.0 SCOPE:     This document applies to determination of CFU-F in non-expanded cultured human bone marrow

3.0 RESPONSIBILITY: All BSR personnel are responsible for following this SOP

 

4.0 ASSOCIATED DOCUMENTATION:

Percutaneous Autologous Bone-Marrow Grafting for Nonnunions.  Influence of the Number and Concentration of Progenitor Cells.  Hernigou et. al   J. Bone Joint Sug Am 87:1430-1437, 2005.

 

5.0 PROCEDURE:

5.1 Materials 5.1.1 T-25 tissue culture flasks

5.1.2    Mesenchymal Stem Cell Basal Media  Poietics Cat No. PT-3238

5.1.3    L-Gltamine PT 4107E

5.1.4    Mesenchymal Stem Cell Growth Suplement PT-4108E

5.1.5    Pent-Step PT-4105

5.1.6    Prepare complete media as per manufacturers instructions

 

5.2 Equipment

 

5.2.1 Incubator, 37 deg C and 5% CO2

5.2.2  BioSafety Hood

 

5.3 Cultures 5.3.1 Seed each sample into three flasks at 2x106 cells in 10ml complete media

5.3.2 Culture for three days

5.3.3 Remove media and non-adherent cells and add fresh complete media

5.3.4      Change media each 3 days until day 10-14.

5.3.5      Stained flasks with Giemsa and analyze with an inverted microscope and 25X objective

5.3.6      An aggregate of cells containing more than thirty cells is counted as a colony.

 

5.4 Results

 

5.4.1 The mean number of fibroblast colony-forming units per 2x106  cells is reported

 

6.0 REVISION HISTORY

 

Revision # Description of Change
New New Document

 

PATIENT     SELECTION

 

 INCLUSION  CRITERIA:

 

  • Patients < 70 years old.
  • Coronary artery disease with an open infarct- related artery
  • A myocardial infarction which at time of transplant therapy later that twelve weeks old.
  • Sinus  rhythm
  • Clear- cut demarcation of the ventriculographic infarcts area.
  • Patients who do not demostrate organ disease.
  • Stable ventricular dynamics for infarct size.
  • Stable ejection fraction
  • Wall movement velocity of infarcted area of at least 9 +/- 6months prior to cell therapy
  • Infarct size at time of cell therapy showed an amount of 27 +/- 8% of the circumference of the left ventricle as determined by ventriculography.

 

CRITERIA TO EXCLUDE A PATIENT FROM THE PROCEDURE

 

  • History of myocardial infarction twelve weeks previous to cellular cardiomyoplasty
  • Previous history of tachycardia or ventricular fibrillation.
  • History of fainting during the past year.
  • Presence of implanted cardiac defibrillator
  • History of active neoplasm or previous chemotherapy treatment.
  • PATIENTS with  DIABETES
  • Myopathy, a history of muscular pathology
  • The patient should not have a grave concomitant or uncontrolled pathology such as chronic renal insufficiency, poorly handled liver disease or uncontrolled hypertension.
  • Patients who can not fulfill the requirements of the protocol because of geographic location, psychiatric or social situations.

 

CLINICAL EVALUATION, LABORATORY ANALYSIS AND FOLLOW UP

 

SCREENING.

 

Initial screening will include the following:
Medical History

 

General evaluation of patient´s health.

 

Physical examination including complete exploration ,cardiac frequency.

Blood pressure and temperature.

Height and weight.

Evaluation of cardiac symptoms and scale of function according to the New York Heart Association.

Additional tests including radiology to determine pathology and general condition.

  1. Electrocardiogram
  2. Chest  X –Ray
  3. Rest and stress (associated with physical exercise) Echocardiogram

Evaluation of global and segmental systemic function by measuring ejection fractions and ventricular diameters, mitral ring movement and regional mobility with necessary techniques. The stress test will serve to estimate functional capacity.

It should be done with a ciclo-ergometer, increasing the inictial charge of 15 volts by 15. The total length of exerceise, level reached and an estimate of functional capacity should be measured.

  1. Magnetic Resonance Imagen to determine cardiac function,      regional motility, myocardial thickness, diameters and volume.
  2. Positron Emission Tomography amount of necrotic myocardial tissue present.
  3. Electrocardiogram for 24hours to evaluate arrythmias, fundammentaly ventricualr in nature.
  4. Coronariography Cardiac Catheterization to identify lesions and plan procedure for revascularization.

Laboratory:

  1. Hematological studies,  CBC, platelets…
  2.  Serum bio-chemistry, billirubin, creatinine, alkaline phosphatase,

Albumin, ALT, AST, and LDH.

  1. Cardiac enzymes, CPK-MB, Troponine.
  2. Serology: HIV, VH-B, VH-C,  IgM and Ig G, anti CMV, Shyphilis, Human T-Cell Leukemia Virus.
  3. P.C.R.
  4. BNP, and Cardiotrophine 1 for labaratory evaluation of IC (23,24) grade.
  5. Peptide Fibrosis markers and apoptosis  (PIP, CTTP y Anexina)

 

BONE  MARROW  STEM  CELL TRANSPLANT  AND REVASCULARIZATION PROCEDURE.

 

Using the Seldinger technique a transfemoral cardiac catheterization  using a number JL 7Fr cayheter guide is introduced into the ostium of the left coronary artery and a routine coronariography is carried out in diverse proiections.   A  dose of  100 UI /  Kg of heparin is administered to keep coagulation time two to three times higher than a control time and the patient is monitored for 30 minutes.

Once the damaged artery has been localized, by using   coronary angiography +  left ventricular injection  ,  a ballon catheter is inserted  using a catheter guide in order to prevent any reverse flow of the cells and at the same time to allow for high pressure injection of the cells into the infarct zone. This way contact time for the cellular migration to the damaged area is prolonged . Four to six doses of 2 to 3 mls. Of cells are introduced low 2 to 4 atms.. Each injection contains between 1.5 to 4.0 x 10 6 of bone marrow cells. The catheters are removed and the patient is monitored in the UCI for the next 12 hours.

 

FOLLOW  UP   EVALUATION.

 

Following the bone marrow transplant and revascularization the patient is transferred to the Intermedial Care Unit  for monitoring. Transfer to the hospital floor subsequent discharge will be carried out according to standard criteria for this type of patient.  The controls to be used following the transplant procedure are the following :

 

While in the hospital:

  • Continuous monitoring to detect and evaluate any cardiac arrhtmias.
  • Laboratory analysis
  • Cardiac enzymes every six hours until stable.
  • Blood work every 72 hours that include reenal and liver function
  • ECG and chest X-ray per order of staff physician.

 

Before discharge :

  • 12 lead ECG
  • Echocardiogram
  • ECG holter

 

After discharge:

 

The following studies will be carried out in order to evaluate capacity of the autologous myoblasts to implant themselves in the myocardium using a PET scan.

Echocardiogram , magnetic resonance and laboratory tests will also be done in order to determine cardiac function and the patient´s general well being.

 

Three Months After Discharge:

Physical examination and clinical history

Laboratory work: CBC, liver and renal function tests and cardiac enzymes.

BNP, cardiothropin, Peptids fibrosis markers and Apoptosis, PIP, CITP and Anexin V.

ECG           24h Holter           Echocardiogram in rest and stress

Positron Emission Tomography  ( PET)

Magnetic Resonance Image

Coronary Cathetherization and Left Ventriculography.

Ten days later implant of bone marrow stem cells

 

Six Months After Discharge

Physical examination and clinical history

Laboratory work: CBC, liver and renal function tests and cardiac enzymes.

BNP, cardiothropin, Peptids fibrosis markers and Apoptosis, PIP, CITP and Anexin V.

ECG           24h Holter           Echocardiogram in rest and stress

Positron Emission Tomography  ( PET)

Magnetic Resonance Image

 

Twelve Months After Discharge Final Evaluation.

 

Physical examination  and clinical history

Laboratory work: CBC, liver and renal function tests, cardiac enzymes

Cerebral Peptid Natriuretic, Cardiothropine, Peptid fibrosis markers and apoptosis, PIP- CITP  and anexin V.

ECG

24h  Holter  ECG.

Base & Stress Echocardiogram

MRI

PET  Viability of MCS  after 12 months.

 

STATISTICS.

 

Because of the status as a safety and feasibility clinical trial, this investigation will be comprised of twenty  patients who will be treated and ten  control (placebo) patients followed by 3 months. A small but representative number of patients is recommended. Every patient who has received a CMM transplant will be included in the safety and feasibility study.

The global survival rate will be evaluated by following the patient from pre- treatment date to date of death by any cause. The global survival rate will be determined using the Kaplan Meier Method.

 

 

EVALUATION CRITERIA OF THE PATIENTS  RESPONSE TO TREATMENT.

 

Echo-cardiograph: baseline and  stress?

A baseline  echocardiograph will permit us to document the segmentary contractibility of necrotic zones. Viability of myocardial cells can be determined by ecocardiograph using stress protocols even through other more reliable techniques exist ssuch as the PET. Given the streightforwardness of this technique it is used to closely monitor the patient.

Whenever possible possible the stress test should include the maximum amount of  physical exertion possible in order to render the most information. Should a patient be unable to stand physical exertion, the habitual amount of dobutamine, 5—10---20—30—40mg /Kg/min, can be used.

PET –18 Desoxiglucose  (18 FDG) and 14—13 Amonium.

 

Tomography by Positron Emission (PET) is the non- invasive diagnostic imagery technique that quantitative date in absolute perfusion values and myocardial metabolism.  The N- 13 Amonium is a     radio-tracer  which allows us to quantify in absolute values myocardial perfusion during a dynamic PET and the use of a kinetic model. P-18 FDG, is a glucose analogous that completes with it in the transport and inter- cellular phosphorilation. It m=remains trapped in the interior of the myocite because it does not participate in neither the glycolysis nor in the synthesis of gluocogen because the Glucose-6-Phosphate is slow in dephosphorilation.

Because of, the kinetic model of the F-18- FDG obtained by a dynamic PET study, permits the non-invasive quantification of myocardial glucose capture. In ischemic areas,blood flow is reduced and energy consummation preferably comes from anaerobic glucolysis. Because of this, there is a reduced capture of N-13-Amonium and an increase in the capture of F-18-FDG in respect normally perfused tissue with which the quantification of myocardial flow and tissue consummation of glucose allows for the tissue to be identified. Necrotic areas are not incorporated into the N- 13 Amonium and F 18 FDG tracers.

 

MAGNETIC  RESONANCE.

Magnetic Cardio Resonance provides us with the means to measure  exactly and observe with little variability, internally distinct cardiac parameters  of great interest to this research.   The capacity to measure cardiac diameters and volumes, parietal thickness, segmentary mobility and global systolic function stands out.  A myocardial viability study using and MCR is based on anatomical evaluation of the function, metabolism and latent hyper-capture of contrast material.  To do this, sequences that study the left ventricle at the base line on long and short axis planes can be  used.  These sequences evaluate by segments the thickness of the walls as well as their movement.  The MCR permits evaluation of these parameters much more precisely than other imagery techniques such as echocardiography.  When debutamine at low doses, 5-10 mg/kg/min, an MCR will demonstrate SSFP sequences, evaluate contractile response to an inotropic stimulus.  Segments whose thickness and mobility have improved with low doses of dobutamine are considered viable.  Once again, the image quality of an MCR as well as its independence from the patients acoustic conditions make it the  ideal imagery for this type of study.  Finally, the slow hyper-capture of Gadolinio contrast is evaluated, a factor which has recently proven to be a sensitive and specific indicator of the absence of viability.  With its high spatial resolution the MCR permits an anatomic, functional, metabolic and perfusion study  with which to obtain definite data on the presence of viable myocardium.  The MCR also determines absolutely systolic function and ventricular diameters in an absolutely reliable manner..

 

TOXICITY AND ADVERSE EFFECTS

 

All adverse effects will be recorded in a report kept on each patient.  The physician will determine if these effects are related to medication, not related, improbable, possible, probable, definitely or unable to evaluate, and their decision will be recorded on the adverse reaction sheet in each patients chart.  Not to be considered secondary effects or toxicity are those adverse reactions unrelated to the medication.  That is to say, they will be reported as not related or relation improbable and will be recorded on separate forms.

 

Definitions:

Adverse Effect:  AE is defined as any occurrence or unfavorable medical experience of a patient or subject in a clinical trial that occurs after the administration of any medication related to the study, not withstanding dose or unofficial connection.  This may include symptoms such as skin rash, enlarged liver, nausea, chest pain, abnormal laboratory findings which include blood work, imagery or scanners or any temporary malaise associated with the use research medication.

 

Adverse reaction to medication:  This can be defined as any negative or unexpected reaction to a medication in any dose.  The causal relationship between any medication and an adverse effect is possible and must not be ignored.

 

Grave Adverse Effect:  This is defined as any undesired experience affecting a patient related or not to research protocol.  A grave adverse effect considered relative to protocol treatment will be defined as Grave Adverse Effect to Medication.  Grave adverse effects and grave adverse effects to medication are those which give rise to:

-Death

-Immediate danger of death to the patient during time reaction is observed.

-Hospital stay or prolonged hospital stay

-Persistent or significant incapacity

-Congenital anomaly or birth defect

-Any other important medical condition.  Important adverse reactions  that do not appear to be life threatening or worthy of hospitalization but that put the patient in danger or may require intervention to prevent any of the above mentioned risks.

 

Toxic death is defined as death associated with toxicity.  This must be specified on the death certificate:   “death due to toxicity”.  Evaluation of toxic death is independent of the hypothesis:  “patients may die from toxic mortality after a complete evaluation of the response to treatment has been made.

 

GRAVE ADVERSE EFFECTS WILL BE REPORTED IMMEDIATELY ACCORDING TO PROCEDURE AS STATED IN THIS PROTOCOL

 

Records and reports of adverse effects.

Adverse effects which are not considered grave and adverse medication effects which are not considered grave.

These must be recorded in the appropriate forms.  All adverse effects and adverse effects due to medication that take place during time of treatment or within 30 days following the administration of the last dose of treatment must be recorded.  The investigator will decide if these effects are related to medication (not related, improbable, possible, probable, definite or unable to evaluate), and his decision will figure in the toxicity formula.  Adverse effects which are definitely not related with the medication (that is reported as not related), will not be considered as adverse effects due to medication in the toxicity report and will be independently reported.

 

GRAVE ADVERSE EFFECT EPISODES AND GRAVE ADVERSE EFFECT EPISODES DUE TO MEDICATION:

 

All grave adverse effect episodes which occur during the treatment or in the 30 days following administration of the last dose of treatment following the protocol must be reported to the lead investigator and to the pharmaceutical firm responsible for the medication whether they are related or not to the study.  The episode will be reported to the proper authorities.

A report of the adverse episode must be made by fax within the first 24 hours after the initial observation of the episode.  Details of the episode must be documented in the form designated for adverse effect episode.

To allow the investigator to fulfill the required information form, complete documentation of all grave adverse effect episodes and grave adverse effect episodes due to medication must be remitted in a period not to exceed 10 days  from the initial report.

It must be noted that adverse effect episodes and adverse effect episodes due to medication which have not been previously documented in the Research Document or that occur in a graver form than anticipated (unexpected), will be subject to a rapid communiqué to the regulatory authorities by the Chief Investigator.  This procedure will also apply to information received from spontaneous sources and any other type of clinical research or epidemiology, independent of its purpose or design.  The source of information should always be specified.

 

ALL ADVERSE EFFECTS REPORTS MUST BE SIGNED AND TIME NOTED BY THE PRINCIPAL RESEARCHER OR OTHER AUTHORIZED PERSONNEL

 

Evaluation of cause and severity of an adverse effect.

The investigator will  evaluate the cause of the following definitions:

 

TERMINOLOGY DEFINITION
Unrelated No evidence of any reaction
Improbable There is little evidence to suggest cause for a reaction. For example: the event was not reported within a reasonable period of time after administration of any medication used in the study; patient’s clinical condition or other ongoing treatments.
Possible

 

Evidence exists that suggest there could be a possible causal reaction due to the fact that the incident occurred within a reasonable time after the administration of medication used in the study.  However, other factors may have contributed to the incident, for example, the patient’s clinical condition, other ongoing treatment.
Problable Evidence exists that suggests a causal relationship to treatment medication and other influencing factors are unlikely.
Definite Clear evidence exists that suggest a casual relatioship, other possible influencing factor can be eliminated
Unable To Evaluate There is insufficient or incomplete evidence to establish a clinical judgement of the cause

 

 

 

INTENSITY DEFINITION
Minimum Adverse effect is detected but does not interfere with patients life.
Moderate Adeverse effect cause discomfort and interferes with patients life.
Severe Adverse effect severely limits patients capacity to carry out day to day activities and requires.
Life Theatening Adverse effect directly puts patients life in danger.

 

 

 

 

ETHICAL ASPECTS

 

General Considerations

 

The trial will be carried out in accordance with the requirements laid out in the Helsinki Document (Edinburgh Revision of October, 2000), and heeding recommendations of the General Health Law (Ley General de Salud), which has established requirements concerning medication trials.  The protocol will be submitted to the Ethical Committee of each institution where the procedure could be carried out.  The trial will begin only after all requirements of the competent convening authorities have been met.

 

INFORMATION THAT WILL BE GIVEN TO TRIAL SUBJECTS AND CONSENT FORMS THAT WILL BE NECESSARY TO PERFORM THE TRIAL.

 

Each person asked to participate in the clinical trial will be given a written document entitled  “Information for Patients”, which will contain information on the following aspects of the clinical trial.

 

 

  • Objective
  • Methodology
  • Treatment patient can expect
  • Benefits that may be obtained for the patient and society.
  • Discomfort and risks the clinical trial may produce such as: frequent         appointments, complimentary tests.
  • The possibility of the occurrence of adverse effects.
  • Other available treatment.
  • The voluntary character of person’s participation in the clinical trial as well as their right to retire from same at any time.  This will in no way compromise their doctor-patient relationship or influence their treatment.
  • Who will have access to patient’s confidential information and how this information will be protected.

 

The chief investigator is responsible for the trial and to adequately answer all  the patient’s questions, doubts and who to contact in case of an emergency.  In accordance with existing law, the chief investigator will obtain the necessary permit from the patient or that persons legal guardian. The patient will give written consent that they are participating under their own will and responsibility.

The participant or his/her representative can at any time revoke the permission without the need to give a reason and in no way will be held responsible.

 

ACCESIBILITY OF DATA TO VOLUNTEERS:

In order to guarantee the confidentiality of the clinical trial results, only the chief investigator and his team of collaborators, the Clinical Research Ethics Committee of the hospital where the trial is taking place and pertinent health authorities.

 

PROTECTION OF INFORMATION OBTAINED FROM CLINICAL TRIAL:

Contents of records or charts that contain information obtained from the clinical trial as well as any documents generated by the study will be protected from use by any person or persons not directly associated with the clinical trial.  They will be considered strictly confidential and the contents will not be revealed to any third party except under the aforementioned specifications.

 

LEGAL RESPONSIBILITY:

            An insurance policy or undersigned civil indemnity and characteristic of the same will be attached.

 

RESPONSIBILITY OF CHIEF CLINICAL TRIAL RESEACHER:

  • Sign protocol of clinical trial
  • Have a total understanding of properties of medication to be used.
  • Obtain signed consent form from patient before their inclusion in trial.
  • Collect, register and communicate to pertinent personnel all data in the correct form.
  • Immediate notification of any grave or unexpected adverse effects.
  • Guarantee that all persons implicated in the clinical study respect the confidentiality of the volunteers.
  • Provide regular information to the Ethics Committee as clinical trial progresses.
  • Take responsibility for and approval of the collection of data at the end of the clinical trial thereby approving it.

 

RULES REGARDING KEEPING OF DATA COLLECTION BOOK:

 

A data collection book will be used in this clinical trial with specifically designed pages for each researcher to fill in.

 

ALL CLINICAL RESEARCHERS AGREE TO:

 

  • Register the patient as soon as he/she has been accepted in the clinical trial.
  • Fill out forms in said book with facts that pertain to each patient.
  • When patient is registered
  • Preliminary examination
  • Follow-up examination
  • The following should be observed when filling out forms:
  • Forms should be signed by chief investigator.
  • All areas must be filled in; if information is lacking place the letters NA (not available) in the corresponding area.
  • Any untoward or extreme results  or those that do not correspond to the expected sequence should be proven.  Any corrections made require the initials, signature, date and brief explanation of the situation.
  • Laboratory results which exceed normal expectations established by the center’s laboratory should be proven by the researcher and a note should be made of them which is then initialed and signed.The normal range of all          laboratory analyses of the center should be made available to the chief           investigator.
  • All results and annotations must be legible if not, the material can be considered lost.
  • The researcher will make a final revision of each and every one of the Data Collection Books and if he approves will sign it.

 

PROTECTION OF CLASSIFIED CLINICAL DATA:

 

Contents of the DCB and any other documents pertinent to the study will be strictly confidential and not be provided to any third party.  Anonymity of the trial subjects will be maintained at all times. Results and conclusions from the clinical trial will be published in medical or scientific journals before this information is released to the general public. Said information will not be made known in a premature or sensationalist manner.