Patent Publication Number: US-2017354693-A1

Title: Cell-based composition and use thereof for treatment of macular oedema and degeneration

Description:
CROSS-REFERENCE TO RELATED APPLICATIONS 
     The present application claims the benefit of Malaysian Application No. PI 2016702121, filed on Jun. 9, 2016, which is hereby incorporated by reference. 
     FIELD OF THE INVENTION 
     The present invention relates to a cell-based composition and use thereof for treatment of complications arising from diabetic maculopathy, more specifically, macular oedema and degeneration. 
     BACKGROUND OF THE INVENTION 
     Diabetes mellitus is a debilitating disease which affects 180 million people worldwide. It is can be classified into two categories, type I diabetes which is previously known as insulin dependent or juvenile onsel, and type II diabetes, also known previously known as non-insulin dependent or adult onset. 
     One of the clinical manifestations resulting from this disease is diabetic maculopathy, particularly macular oedema which affects loss of vision. Maculopathy can be classified into several categories namely focal, diffuse, ischaemic, tractional or a combination thereof. It can considered as clinically significant if the oedema affects the macular region in accordance to Early Treatment Diabetic Retinopathy Study (EDTRS). Macular oedema is the leading cause of blindness associated with diabetes. It is characterised by thickening and swelling of centre of the retina resulting from accumulation of excess fluid in outer plexiform layer and inner nuclear layer of the eye which causes deterioration of the eye vision dramatically. Among those who are particularly prone to develop diabetic macular oedema include poorly controlled diabetics, patients with hypertension and prolonged diabetics. 
     In efforts to treat diabetic maculopathy, various treatment methods have been developed in the past. Conventionally, management of diabetic maculopathy is carried out by means of laser treatment. Studies have shown that patients with clinically significant macular oedema who had undergone argon laser treatment resulted in lower incidence of blindness compared to control. Generally, laser treatment do not improve the vision although it may reduce the loss of vision by around 50% within three years. 
     In recent years, there has been a growing trend to use anti-vascular endothelial growth factor, or commonly known as anti-VEGF, to reduce occurrence of macular oedema. This form of therapy has now replaced laser treatment as it has produced results in better visual acuity as compared to argon laser treatment. However, anti-VEGF therapy requires a drug to be delivered intra-vitreally. Furthermore, multiple injections into the eye are is required to achieve favourable results. Typically, injection intervals are required in repetition at least once in every one or two months which is a rather burdensome procedure. Hence, this is not cost-effective nor economically favourable to the patients. Another drawback arising from anti-VEGF therapy is a myriad of adverse complications which include cataract, vitreous haemorrhage, endophthalmitis and retinal detachment. 
     In light of the above, medical experts have begun to look into other ways for treating diabetic maculopathy which are safer, non-invasive and clinically effective. Studies on mesenchymal stem cells, also known as mesenchymal stromal cells, have generated a lot of interest among researchers and clinicians due to its attributes which include regenerative properties and immunomodulatory capacities. Kavanagh et al. (2015) has reported on therapy using mesenchymal stem cells and its effects in preventing allergic airway inflammation on ovalbumin (OVA) sensitized mice. It was shown that this therapy was able to reduce allergen-driven eosinophilia and suppress allergen-specific immunoglobulin E (IgE) response. A significant reduction in levels of interleukin 4 (IL-4) and interleukin 13 (IL-13) was found but a marked increase in interleukin 10 (IL-10) inducing a regulatory T-cell population in OVA sensitized mice which were treated with mesenchymal stem cells. This illustrates that the protective effect of mesenchymal stem cells was a result of a targeted, specific immunomodulation rather than a global suppression of the immune response. 
     However, studies and research publications on treating macular oedema and degeneration in humans using cell-based methods appears to be lacking. Accordingly, there remains a need for a novel cell-based composition which is clinically safe and therapeutically effective for treatment of macular oedema and degeneration in humans. 
     SUMMARY OF THE INVENTION 
     In overcoming the above challenges resulting from conventional methods in the past, the present invention provides a cell-based preparation and use thereof which is clinically safe and therapeutically effective for treatment of diabetic maculopathy in humans. 
     More particularly, the present invention relates to a cell-based composition comprising a suspension of mesenchymal stem cells in crystalloid with a cellular concentration from 0.01 million to 3.0 million cells/ml wherein the cell-based composition is used in a form of medicament for treatment of macular oedema and degeneration. 
     Thus, one aspect of this invention is a method for treating macular oedema and degeneration in a subject by administering to the subject (e.g., by intravenous infusion carried out for 0.5 to 2 hours) the cell-based composition described above and hereinafter in a therapeutically effective amount (e.g., 0.25 million to 3.0 million cells/kg body weight). 
    
    
     
       BRIEF DESCRIPTION OF DRAWINGS 
       The drawings constitute part of this specification and include an exemplary or preferred embodiment of the invention, which may be embodied in various forms. It should be understood, however, that the disclosed preferred embodiments are merely exemplary of the invention. Therefore the figures disclosed herein are not to be interpreted as limiting, but merely as the basis for teaching one skilled in the art of the invention. 
       In the appended drawings: 
         FIG. 1  illustrates morphology of human cord-derived mesenchymal stem cells. 
         FIG. 2  illustrates immunophenotyping assay results of the mesenchymal stem cells. 
         FIG. 3  illustrates adipogenesis, osteogenesis and chondrogenesis of the mesenchymal stem cells respectively. 
         FIG. 4  illustrates significant reduction in central macular nerve fibre thickness of a patient A, corresponds to the area of macular oedema following the intravenous injection of the cell-based composition. 
         FIG. 5  illustrates significant improvement in central macular nerve fibre thickness of a patient B following intravenous injection of the cell-based composition. 
         FIG. 6  illustrates improvement on patient A&#39;s visual acuity from 6/24 to 6/12 following initial treatment using the cell-based composition. 
         FIG. 7  illustrates significant reduction in macular oedema coupled with significant improvement in vision from 6/12 to 6/6 after 10 months and 6/7.5 at 17 month as a result of patient B receiving a single injection of the cell-based composition injections. 
         FIG. 8  illustrates optical coherence tomography results for patient A exhibiting recovery of retinal macular oedema after 8 months from treatment using the cell-based composition. 
         FIG. 9  illustrates optical coherence tomography results for patient B exhibiting recovery of retinal macular oedema after 6 months from treatment using the cell-based composition. 
     
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     A detailed description of the present invention is described herein. The present invention is directed to a cell-based composition and use thereof for treating complications arising from diabetic maculopathy. More particularly, the present invention relates to a cell-based composition comprising a suspension of mesenchymal stem cells in crystalloid with a cellular concentration from 0.01 million to 3.0 million cells/ml wherein the cell-based composition is used in a form of medicament for treatment of macular oedema and degeneration. 
     Accordingly, the mesenchymal stem cells used for preparation of the suspension are derived from various sources including, but not limiting to, human umbilical cord, bone marrow, fat tissue, peripheral blood or tooth pulp. Samples are either collected from mothers post-birth or from healthy donors. If required, the samples are cleaned and disinfected accordingly. 
     Upon collection, the samples are sent to laboratory to be processed further. The samples will be digested using a digestion enzyme, preferably, but not limiting to collagenase type II and followed by centrifugation, leaving a layer of supernatant and pellet containing mesenchymal stem cells. The mesenchymal stem cells are isolated and cultured in a specially formulated medium supplemented with a combination of various antibiotics and animal-free serum. The cultures are maintained at a temperature range from 35° C. to 40° C., preferably at 37° C. in a humidified atmosphere for 3-4 days. 
     As it will be apparent to a person of ordinary skill in the art, mesenchymal stem cells are adherent to plastic. Non-adherent cells are discarded and the growth medium is replaced every 3-4 days until the cells reached confluence. 
     Upon reaching 70%-80% confluence, the adherent mesenchymal stem cells are incubated with a dissociation enzyme, preferably, but not limiting to trypsin and re-plated at 1×10 4  cells/ml for a series of passages, preferably, but not limiting to 3-4 passages. The mesenchymal stem cells are then harvested in a culture flasks, thus expanding population of the cells. 
     The mesenchymal stem cells are characterized in accordance to a criteria set forth by International Society for Cellular Therapy (ISCT). Apart from adherence to plastic, established criteria defining mesenchymal stem cells include expression of antigen markers as measured by flow cytometry and tri-differentiation ability of the cells (Dominici 2006). Using flow cytometry, the mesenchymal stem cells are defined by expression of CD73, CD90, and CD105 markers whilst absence of expression for CD34, CD45, and HLA-DR markers. Meanwhile, the tri-differentiation ability of the mesenchymal stem cells is demonstrated by way of the cells differentiating into osteoblasts, adipocytes and chondroblasts. 
     Once the population of mesenchymal stem cells have been expanded, a disassociation enzyme, preferably, but not limiting to trypsin is added in to the flasks and incubated at a temperature range from 35° C. to 40° C., more preferably at 37° C., for a period of 1-15 minutes, more preferably for 5 minutes to detach the plastic-adherent mesenchymal stem cells, leaving the cells slightly shrunk. Next, the flasks are gently tapped to dislodge the cells and medium is further added to dilute the trypsin, forming a mesenchymal cell suspension. The cell suspension is then transferred into 50 ml centrifuge tubes and centrifuged at a speed range from 300 g to 800 g, more preferably at 500 g at a temperature range from 18° C. to 20° C. for 10 minutes forming a layer of supernatant with pellet at bottom of the tubes. The supernatant is removed, leaving the pellet of mesenchymal stem cells in the tubes. 
     The pellet of mesenchymal stem cells are re-suspended in a sterile cryovial of 1.8 ml in size containing cryopreservation medium comprising from 80% to 90% animal-free serum and a cryoprotectant, preferably, but not limiting to dimethyl sulfoxide from 1% to 10%. Alternatively, dimethyl sulfoxide may also be substituted with human serum albumin. Typically, a cryovial contains from 25 million to 30 million cells per vial. Alternatively, cryovials of up to 5 ml in size may also be used. 
     The mesenchymal stem cells in cryovials are frozen in a controlled rate freezer until −70 to −90° C. but preferably −90 gradually before transferring into quarantine tank, preferably, but not limiting to a vapour phase liquid nitrogen storage tank. 
     To prepare a cell-based composition for the treatment of macular oedema and degeneration, the cryopreserved mesenchymal stem cells in cryovials are first thawed at a temperature ranging from 30° C. to 40° C., preferably at 37° C. in a water bath or an incubator for a period from 1 to 5 minutes, more preferably at 2 minutes. Next, the cells are then transferred into new sterile cryovials and are washed with sterile saline, is preferably but not limiting to 0.9% sodium chloride. The washed cells in the sterile cryovials will then be centrifuged at a speed ranging from 500×g to 1000×g, preferably at 800×g for a period from 3 to 10 minutes, more preferably for 5 minutes at room temperature, forming a layer of supernatant and a pellet of mesenchymal stem cells. 
     Typically, the supernatant is removed and discarded, leaving the pellet of mesenchymal stem cells in the cryovial. The pellet of mesenchymal stem cells are then re-suspended with sterile saline at a volume ranging from 5 to 20 ml, preferably at 10 ml, forming a suspension of mesenchymal stem cells. 
     A cell-based composition is then prepared by infusing the suspension of mesenchymal stem cells into crystalloid to reach a cellular concentration from 0.01 million to 3.0 million cells/ml. The crystalloid includes, but not limiting to normal or half-normal saline or colloid. 
     Exact amount of cells per kg body weight to be administered into a patient depends on variety of factors including body weight, route of administration, age and gender of the patient, and also the type of mechanism of action targeted. Typically, the therapeutically effective amount of the cell-based composition used for the treatment of macular oedema and degeneration is from to 0.25 million to 3.0 million cells/kg body weight. 
     The following examples further illustrate but by no means limit the scope of the invention: 
     Example 1: Collection and Handling of Umbilical Cord Sample 
     The umbilical cord sample was detached from placenta of a donor post-birth using medical scissors and was immediately submerged in povidone iodine solution for 1-5 minutes to eliminate bacteria and to avoid any risk of contamination. Alternatively, the is umbilical may be disinfected using alcohol swab. Upon disinfection, the umbilical cord was then placed in a sterile container of sterile saline solution to maintain moisture. Subsequently, the sterile container was placed into a collection kit and was transported to laboratory using a thermo-insulated bag and kept under a temperature range from 4° C. to 37° C. 
     The sample was then processed within 48 hours from time of collection. 
     Example 2: Isolation and Culture of Mesenchymal Stem Cells 
     First, veins and arteries of the umbilical cord were removed and followed by mincing into 1-2 mm fragments. The fragments were digested with an enzyme, preferably, but not limiting to 0.01% to 0.05% collagenase type II, for a period from 1 to 3 hours, forming a mixture. Next, a centrifugation was carried out to separate the mesenchymal stem cells from the mixture. The mesenchymal stem cells were isolated and then cultured in a growth medium, preferably, but not limiting to Dulbecco&#39;s Modified Eagle&#39;s Medium (DMEM) which may or may not contain low glucose supplemented with 5-20% animal-free serum and a combination of antibiotics comprising 100 U/mL penicillin, 100 mg/mL streptomycin, 250 ng/mL amphotericin B and 2 mM glutamine. The cultures were maintained at 37° C. in a humidified atmosphere of 5% CO 2  and 95% air for 3 days. 
     Non-adherent cells were discarded and the growth medium was replaced every 3-4 days until the cells reached confluence. 
     Next, the plastic-adherent mesenchymal stem cells were incubated with trypsin and re-plated at 1×10 4  cells/ml for 3-4 passages. The mesenchymal stem cells were then harvested in a culture flasks, thus expanding population of the cells. 
       FIG. 1  illustrates the morphology of the mesenchymal stem cells. 
     Example 3: Characterization of Mesenchymal Stem Cells 
     Immunophenotyping 
     The mesenchymal stem cells were immunophenotyped at passage three using isotype (fluorescein isothiocyanate) FITC and (phycoerythrin) PE controls with antigen markers which include CD34, CD45, CD73, CD90, CD105 and HLA-DR. As shown in  FIG. 2 , the immunophenotyping assay results for the mesenchymal stem cells validate expression for CD73, CD90 and CD105 whilst lacking expression for CD34, CD45 and HLA-DR. 
     Differentiation Assay 
     To perform this assay, a selection of specially formulated differentiation medium were used to induce tri-differentiation ability of the mesenchymal stem cells. 
     Adipogenesis: 
     The mesenchymal stem cells were treated in adipogenic differentiation medium comprising complete medium supplemented with 1 mM dexamethasone and 0.2 mM indomethacin, 0.01 mg/mL insulin and 0.5 mM 3-isobutil-1-metil-xantina. The medium was replaced every 3 days, and the differentiated cells were subjected to Oil Red 0 staining after about 14 days of culture. 
     Chondrogenesis: 
     The mesenchymal stem cells were cultured in pellet form and maintained in a chemically defined basal medium comprising complete medium supplemented with 50 mg/mL ascorbate-2-phosphate, 1.0 mM sodium piruvate, 40 mg/mL proline, 10 ng/mL transforming growth factor-b3, 6.25 mg/mL human insulin, 6.25 mg/mL transferrin, 6.25 mg/mL bovine insulin, 6.25 mg/mL selenous acid, 1.25 mg/mL linoleic acid, and 5.35 mg/mL bovine serum albumin. Next, the cells were suspended in 1 mL of chondrogenic medium and replaced every 3-4 days. Chondrogenic pellets were harvested after 5 weeks in culture. To assess chondrogenesis, Alcian Blue was used to stain cartilage matrix. 
     Osteogenesis: 
     The mesenchymal stem cells were treated in osteogenic differentiation is medium comprising complete medium supplemented with 50 mg/mL ascorbate-2-phosphate, 10 mM b-glycerophosphate, and 100 nM dexamethasone. The medium was replaced every 3 days continuously for 2-3 weeks. Alizarin Red S was used to stain matrix mineralization associated with differentiated osteocytes. 
       FIG. 3  demonstrates the tri-differentiation ability of the mesenchymal stem cells exhibiting adipogenesis, osteogenesis and chondrogenesis respectively. 
     Example 4: Cryopreservation of Mesenchymal Stem Cells 
     Once the population of mesenchymal stem cells was expanded, trypsin was added in to the flasks and incubated at 37° C., for 5 minutes to detach the plastic-adherent mesenchymal stem cells, leaving the cells slightly shrunk. Next, the flasks were gently tapped to dislodge the cells and medium was further added to dilute the trypsin, forming a mesenchymal cell suspension. The cell suspension was then transferred into 50 ml centrifuge tubes and centrifuged at 500 g at a temperature ranging from 18° C. to 30° C. for 10 minutes forming a layer of supernatant with pellet at bottom of the tubes. The supernatant was removed, leaving the pellet of mesenchymal stem cells in the tubes. 
     The pellet of mesenchymal stem cells were re-suspended in a sterile cryovial containing cryopreservation medium comprising up to 90% animal-free serum and up to 10% dimethyl sulfoxide and were cryopreserved in a controlled freezing gradual rate at −90° C. before being transferred into a quarantine tank at −190° C. 
     Example 5: Preparation of the Cell-Based Composition for Treatment 
     The cryopreserved mesenchymal stem cells in cryovials were thawed at 37° C. in a water bath or an incubator for 2 minutes. Next, the cells were then transferred into new sterile cryovials and were washed with 0.9% sodium chloride. The washed cells in the sterile cryovials were then centrifuged at 800×g for 3-10 minutes at room temperature, forming is a layer of supernatant and a pellet of mesenchymal stem cells. The supernatant was removed using a sterile syringe, leaving the pellet of mesenchymal stem cells in the cryovial. 
     The pellet of mesenchymal stem cells was then re-suspended with sterile saline at 10 ml, forming a suspension of mesenchymal stem cells. A cell-based composition was then prepared by infusing the suspension of mesenchymal stem cells into saline at a volume of 250 ml, in a sterile bottle. 
     Example 6: Treatment Procedure Using the Cell-Based Composition 
     Treatment using the cell-based composition was carried out on two patients who were afflicted by macular oedema. Patient A, was a 62-year old male, and patient B, was a 67-year old female. Both patients were diagnosed as diabetic. Patient A was given three injections of the cell-based composition, first at a dose of 100×10 6  cells and at a dose of 75×10 6  cells for second and third injections. Meanwhile, patient B received a single injection dose of 50×10 6  cells. 
     The treatment procedure began with infusion of 200 ml saline into the patients for a period from 45 to 60 minutes. Next, the cell-based composition which was prepared earlier (as described in Example 5) was infused into the patients for a period from 30 minutes to 2 hours. The bottle containing the cell-based composition was shaken gently every 5 minutes to ensure that the cells are suspended in saline homogenously. When the infusion is almost complete, 50 ml of sterile saline was infused into the bottle containing the cell-based composition to rinse and flush out any remaining cells. 
     Example 7: Results and Discussion 
     Following infusion of the cell-based composition, it was found that the macular oedema is in patient A and patient B had resolved significantly. Visual acuity of these patients have also improved. Subjectively, the patients also observed visual improvement. 
     TABLES 1 and 2 below demonstrates the data results of macular thickness and visual acuity of the patients after receiving treatment. 
     
       
         
           
               
             
               
                 TABLE 1 
               
             
            
               
                   
               
               
                 Macular thickness and visual acuity of patient A after treatment 
               
            
           
           
               
               
               
            
               
                   
                 Macular thickness μm 
                 Visual acuity 
               
            
           
           
               
               
               
               
               
            
               
                 Date 
                 Right eye 
                 Left eye 
                 Right eye 
                 Left eye 
               
               
                   
               
               
                 24 Nov. 2014 
                 651 
                 272 
                 — 
                 — 
               
               
                 16 Mar. 2015 
                 553 
                 268 
                 6/24 
                 6/6     
               
            
           
           
               
               
            
               
                 24 Mar. 2015 
                 First injection 
               
            
           
           
               
               
               
               
               
            
               
                 25 May 2015 
                 403 
                 275 
                 6/12 
                 6/7.5 
               
               
                 15 Jun. 2015 
                 544 
                 276 
                 6/15 
                 6/7.5 
               
            
           
           
               
               
            
               
                 27 Jun. 2015 
                 Second injection 
               
            
           
           
               
               
               
               
               
            
               
                 20 Jul. 2015 
                 458 
                 262 
                 6/15 
                 6/7.5 
               
               
                 21 Aug. 2015 
                 340 
                 262 
                 6/30 
                 6/7.5 
               
            
           
           
               
               
            
               
                 24 Nov. 2015 
                 Third injection 
               
            
           
           
               
               
               
               
               
            
               
                 23 Nov. 2015 
                 317 
                 262 
                 — 
                 — 
               
               
                 12 May 2016 
                 261 
                 253 
                 6/12 
                 6/6     
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 2 
               
             
            
               
                   
               
               
                 Macular thickness and visual acuity of patient B after treatment 
               
            
           
           
               
               
               
            
               
                   
                 Macular thickness μm 
                 Visual acuity 
               
            
           
           
               
               
               
               
               
            
               
                 Date 
                 Right eye 
                 Left eye 
                 Right eye 
                 Left eye 
               
               
                   
               
               
                 14 May 2014 
                 374 
                 294 
                 6/12 
                 6/9.5 
               
            
           
           
               
               
            
               
                 24 May 2014 
                 Single injection 
               
            
           
           
               
               
               
               
               
            
               
                 13 Aug. 2014 
                 334 
                 300 
                 6/12 
                 6/7.5 
               
               
                 9 Dec. 2014 
                 292 
                 304 
                  6/9.5 
                 6/9.5 
               
               
                 10 Mar. 2015 
                 299 
                 305 
                 6/6  
                 6/6     
               
               
                 21 Oct. 2015 
                 286 
                 307 
                  6/7.5 
                 6/7.5 
               
               
                   
               
            
           
         
       
     
     As illustrated in  FIG. 4 , significant reduction in central macular nerve fibre thickness was observed in patient A which corresponds to the area of macular oedema following the intravenous injection of the cell-based composition. The oedema recurred around three months later. Following a second dose of injection of the cell-based composition, the macular oedema improves significantly and has remained so till the last follow up to 6 months post-infusion. An improvement in patient A&#39;s visual acuity was observed.  FIG. 6  illustrates improvement on patient&#39;s visual acuity from 6/24 to 6/12 following initial treatment using the cell-based composition. The improvement of visual acuity corresponds with reduction of macular oedema. Two months post-infusion, it was noted that there was a small sub-retinal haemorrhage outside the macular region and the patient subsequently received pan-retinal photocoagulation (PRP). Patient A received a second is injection three months later which resulted an improvement on the macular oedema. 
       FIG. 5  illustrates significant improvement in central macular nerve fibre thickness of a patient B following two cell-based composition injections at three months apart. 
     Improvement on the oedema 17 months following the injections is observed. Further,  FIG. 7  illustrates significant reduction in macular oedema coupled with significant improvement in vision from 6/12 to 6/6 after 10 months and 6/7.5 at 17 month as a result of patient B receiving two injections at three months apart. 
     Results of optical coherence tomography for patient A and patient B exhibiting recovery of retinal macular oedema after 8 and 6 months respectively from treatment using cell-based composition were illustrated in  FIGS. 8-9 . 
     Based on the results from the studies above, intravenous injections of the cell-based composition has shown to significantly reduce the macular oedema related to diabetes. A single injection has led to down immune-modulation of immune system, thus, resulting in significant symptomatic improvement which may improve systemic control of diabetes. Hence, this has brought visual improvement in the patients and eliminated the need for is repeated intravitreal anti-VEGF injection. 
     Having described preferred embodiments of the present invention with reference to the accompanying drawings, it is not intended that these embodiments and examples illustrate and describe all possible forms of the present invention, and it is to be understood that the present invention is not limited to those precise embodiments, and that various changes and modifications may be effected therein by one skilled in the art without departing from the scope of the invention as defined in the appended claims.