# US 2014/0154221 A1 · Treatment method for mesenchymal stem cells and their application as a treatment of oxidative stress related diseases

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- Number: US 2014/0154221 A1
- Document ID: US20140154221A1
- Status: Published application
- Kind: A1
- Application number: 14/131,049
- Filed: 2011-07-06
- Published: 2014-06-05
- Earliest claimed: 2011-07-06
- Assignees: Histocell, S.L.
- Inventors: Maria Begona Castro; Javier Diez Garcia
- Web page: https://patentgrep.com/patent/US20140154221A1

## CPC

- C12N 5/0667: {Adipose-derived stem cells [ADSC]; Adipose stromal stem cells}
- A61P 1/02: Stomatological preparations, e.g. drugs for caries, aphtae, periodontitis
- A61P 1/04: for ulcers, gastritis or reflux esophagitis, e.g. antacids, inhibitors of acid secretion, mucosal protectants
- A61P 3/10: for hyperglycaemia, e.g. antidiabetics
- A61P 9/00: Drugs for disorders of the cardiovascular system
- A61P 9/10: for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
- A61P 11/00: Drugs for disorders of the respiratory system
- A61P 17/02: for treating wounds, ulcers, burns, scars, keloids, or the like
- A61P 25/00: Drugs for disorders of the nervous system
- A61P 25/02: for peripheral neuropathies
- A61P 25/14: for treating abnormal movements, e.g. chorea, dyskinesia
- A61P 25/16: Anti-Parkinson drugs
- A61P 25/28: for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
- A61P 29/00: Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
- A61P 35/00: Antineoplastic agents
- A61P 37/02: Immunomodulators
- A61P 37/06: Immunosuppressants, e.g. drugs for graft rejection
- A61P 39/06: Free radical scavengers or antioxidants
- A61K 35/28: Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
- C12N 2500/02: Atmosphere, e.g. low oxygen conditions
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61K: PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES (devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms A61J3/00; chemical aspects of, or use of materials for deodorisation of air, for disinfection or sterilisation, or for bandages, dressings, absorbent pads or surgical articles A61L; soap compositions C11D)

## Abstract

The present invention relates to a method of treatment of mesenchymal stem cells, preferably from adipose origin, comprising mainly two stages, firstly the collection and isolation of mesenchymal stem cells, and secondly, a period of growth and specific treatment of cells in a conditioning or treatment medium with an oxidizing agent. The invention also comprises the cells obtained directly through the method and its use in treating diseases caused by or associated with oxidative stress.

## Claims

### Claim at position 1

1-17. (canceled)

### Claim 18

A method of treatment of mesenchymal stem cells that comprises obtaining and isolating the mesenchymal stem cells and culturing the cells in a treatment medium comprising an oxidizing agent, characterized in that said culturing of the cells in a treatment medium comprises two consecutive treatment cycles of 48-72 hours, followed by a third pre-conditioning cycle of 24-48 hours in an experimental support.

### Claim 19 (depends on 18)

The method according to claim 18, characterized in that the mesenchymal cells are isolated from adipose tissue, bone marrow, umbilical cord and/or placenta.

### Claim 20 (depends on 18)

The method according to claim 18, characterized in that the mesenchymal cells are isolated from human adipose tissue.

### Claim 21 (depends on 18)

The method according to claim 18, characterized in that the oxidizing agent is selected from the group consisting of: hydrogen peroxide (H2O2), calcium peroxide (CaO2), magnesium peroxide (MgO2), zinc peroxide (ZnO2), manganese peroxide (MnO2), plumb peroxide (PbO2) and nitric oxide (NO), nitrous oxide (N2O), ozone (O3), sodium perborate (NaBO3), selenium dioxide (SeO2), silver oxide (Ag2O), ferric salts such as ferric chloride (FeCl3), copper salts such as sodium percarbonate (2Na2CO3), permanganates such as potassium permanganate (K2Mn2O8), dichromates such as potassium dichromate (K2Cr2O7), lithium, sodium and calcium salts of hypochlorous acid (HClO—), sodium chlorite (NaClO2), chloric acid (HClO3), potassium chlorate (KClO3), aluminium hydroxide (Al2O3), aluminium hydroxide coprecipitated with magnesium carbonate (MgCO3), arsenic trioxide (As(OH)3), benzoyl peroxide ((C6H5CO)2O2), calcium hydroxide (Ca(OH)2), chlordiazepoxide hydrochloride, cupric oxide (CuO), iron oxides, magnesium oxide (MgO), magnesium dioxide, magnesium hydroxide (Mg(OH)2), potassium hydroxide (KOH), sodium hydroxide (NaOH), titanium oxide (TiO2) and/or zinc oxide (ZnO).

### Claim 22 (depends on 21)

The method according to claim 21, characterized in that said culturing of the cells in a treatment medium comprises the following steps:
a) first cycle: seeding the cells in a culture surface and allowing a conditioning time comprised between 4 and 8 hours for the cells to adhere and acquire their typical morphology;
b) adding the treatment medium until reaching a final H2O2 concentration comprised between 0.01 y 0.05 mM;
c) maintaining for 48-72 hours inside the incubator at 37° C. and 5% CO2 atmosphere;
d) second cycle: refreshing the treatment medium until reaching again a final H2O2 concentration comprised between 0.01 y 0.05 mM;
e) incubating these cells for 48-72 hours at 37° C. with 5% CO2;
f) third cycle: refreshing the treatment medium by applying again said medium until reaching once more a final H2O2 concentration comprised between 0.01 y 0.05 mM;
g) incubating these cells for 24-48 hours at 37° C. with 5% CO2.

### Claim 23 (depends on 22)

The method according to claim 22, characterized in that the treatment medium used in steps b), d) and f) comprises conventional cell culture medium at 85-95%, fetal calf serum at 5-15%, antibiotics at 0.5-5% and hydrogen peroxide at 0.01-0.05 mM.

### Claim 24 (depends on 18)

Mesenchymal stem cells directly obtained from the method according to claim 18, characterized in that the cells present an increase of SOD1 gene expression of at least 30% with respect to non treated ASC, measured by PCR method.

### Claim 25 (depends on 24)

The mesenchymal stem cells according to claim 24, characterized in that the cells present an increase of SOD2 gene expression of at least 25% with respect to non treated ASC, measured by PCR method.

### Claim 26 (depends on 25)

The mesenchymal stem cells according to claim 25, characterized in that the cells present an increase of SOD3 gene expression of at least 50% with respect to non treated ASC, measured by PCR method.

### Claim 27 (depends on 26)

The mesenchymal stem cells according to claim 26, characterized in that the cells present an increase of Cat gene expression of at least 50% with respect to non treated ASC, measured by PCR method.

### Claim 28 (depends on 27)

The mesenchymal stem cells according to claim 27, characterized in that the cells present an intracellular increase of GSH of at least of 8% with respect to non treated ASC, measured by Tietze enzymatic method.

### Claim 29 (depends on 28)

The mesenchymal stem cells according to claim 28, characterized in that the cells present a decrease of intracellular levels of MROs of at least 10% with respect to non treated ASC, measured by fluorometric quantitative method with DCFA probe.

### Claim 30 (depends on 29)

The mesenchymal stem cells according to claim 29, characterized in that the cells present an increase of beta-actin of at least 40% with respect to non treated ASC, measured by PCR method.

### Claim 31 (depends on 30)

The mesenchymal stem cells according to claim 30, characterized in that the cells present an increase of IGF-1 of at least 40% with respect to non treated ASC, measured by PCR method.

### Claim 32 (depends on 24)

Use of the cells according to claim 24 for the manufacturing of a medicament for the treatment of diseases related to or caused by oxidative stress and/or degenerative evolution conditions due to oxygen reactive species-mediated compounds.

### Claim 33 (depends on 32)

Use of the cells according to claim 32 for the treatment of diseases selected from the group consisting of: periarteritis nodosa, diabetes mellitus, chronic granulomatous disease, arteriosclerosis, stroke, pulmonary fibrosis (chronic obstructive pulmonary disease, COPD, idiopathic pulmonary fibrosis), ischemia-reperfusion syndrome, Alzheimer's, Parkinson's, rheumatoid arthritis, systemic lupus erythematosus, inflammatory bowel disease: ulcerative colitis and Chron's disease, adult respiratory distress syndrome, atherosclerosis, spinal cord injury, peripheral nerve injury, amyotrophic lateral sclerosis, Huntington's disease, Friedeich's ataxia, periodontitis, mucosal diseases and illnesses and injuries that occur with an inflammation component, acute and chronic ulcers and wounds.