NEW USE OF ANGIOTENSIN II RECEPTOR AGONISTS

FIELD OF THE INVENTION

This invention relates to a new use of compounds that are angiotensin II (Ang II) receptor agonists, more particularly selective agonists of the Ang II type 2 receptor

(hereinafter the AT ₂ receptor, AT2 receptor, AT ₂ R or AT2R), and especially agonists that bind selectively to that receptor, in particular non-peptide selective agonists that bind to that receptor, for use in stem cell and progenitor cell therapy.

BACKGROUND OF THE INVENTION

Renin, a protease, cleaves its only known substrate (angiotensinogen) to form angiotensin I, which in turn serves as substrate for angiotensin converting enzyme (ACE) to form Ang II. The endogenous hormone Ang II is a linear octapeptide (Asp ¹ -Arg ² -Val ³ -Tyr ⁴ - Ile ⁵ -His ⁶ -Pro ⁷ -Phe ⁸ ), and is an active component of the Renin Angiotensin System (RAS). The Ang II type 1 receptor (hereinafter the ATi receptor, ATI receptor, ATiR or AT1R) is expressed in most organs, and is believed to be responsible for the majority of the

pathological effects of excessive Ang II. Several studies in adult individuals demonstrate that, in the modulation of the response following Ang II stimulation, activation of the AT ₂ receptor has opposing effects to those mediated by the ATi receptor.

The expected pharmacological effects of agonism of the AT ₂ receptor are described in general in de Gasparo M et al, Pharmacol Rev 2000; 52: 415-472. More recently, AT ₂ receptor agonists have been shown to be of potential utility in the treatment and/or

prophylaxis of disorders of the alimentary tract, such as dyspepsia and irritable bowel syndrome, as well as multiple organ failure (see international patent application WO

99/43339). AT ₂ receptor agonists have also been described, for instance, in international patent application WO 02/096883. The effects of Ang II on cell growth, inflammation and extracellular matrix synthesis are mainly coupled to ATi, whereas the function of AT ₂ has been heavily investigated and new research indicates that it has properties opposing the ATi receptor. Specific stimulation of the AT ₂ receptor has been shown to be important in anti- inflammation, neuro -protection, neuro-reparation as well as reducing myocyte hypertrophy and fibrosis.

Stem cells have been investigated in several conditions either as a treatment for a specific disease or to improve organ function. However, the therapeutic efficacy of stem cell therapy remains low in many disease conditions, e.g. heart failure after myocardial infarction. Reasons for failed efficacy includes a low proliferation rate of stem cells in cell cultures, inability to fully differentiate to organ specific cells, low survival rate in the recipient, failure to integrate within the targeted tissue, inability to restore organ function, etc.

The AT ₂ receptor is abundant and involved in organ growth and differentiation of more primitive cells into organ specific cells during fetal development. For example, the AT ₂ R appears to be involved in kidney (Norwood et., Am J Physiol 1997; 272: R662-668), (Sanchez et al, Eur J Pharmacol 2008; 588: 114-123) and skin development (Liu et al, J Dermatol Sci 2007; 46: 221-225) during fetal life, supporting the notion that the AT ₂ R may play an important role in organogenesis and organ development. In healthy individuals, AT ₂ receptors are usually present in low numbers but a marked up-regulation of the receptor population is seen in various organs under disease conditions.

SUMMARY OF THE INVENTION

Compounds of the invention are Ang II receptor agonists, more particularly, are agonists of the AT ₂ receptor, and, are selective agonists of that sub-receptor and especially non-peptide selective agonists of that sub-receptor. In some embodiments, the compounds of the invention are those that can stimulate AT ₂ receptors.

Compounds of the invention can therefore be used in stem cell and progenitor cell therapy. In some embodiments, a compound of the invention can be N-butyloxycarbonyl-3-(4- imidazol-l-ylmethylphenyl)-5-z50-butylthiophene-2-sulfonamid e (referred to as Compound 21 or in short C21 herein).

An aspect of the embodiments relates to an agonist of angiotensin II type 2 (AT ₂ ) receptor for use in inducing proliferation and/or differentiation of cells selected from a group consisting of stem cells and progenitor cells.

Another aspect of the embodiments relates to an agonist of AT ₂ receptor for use in inducing mobilization of cells selected from a group consisting of stem cells and progenitor cells into an organ suffering from an injury in a patient.

A further aspect of the embodiments relates to an agonist of AT ₂ receptor for use in pre- treatment of a patient subject to stem cell and/or progenitor cell therapy involving transplantation of stem cells and/or progenitor cells into said patient.

Further aspects of the embodiments relates to a composition comprising an agonist of AT ₂ receptor and an antagonist of angiotensin II type 1 (ATi) receptor and/or an angiotensin converting enzyme (ACE) inhibitor for use in 1) inducing proliferation and/or differentiation of cells selected from a group consisting of stem cells and progenitor cells, 2) inducing mobilization of cells selected from a group consisting of stem cells and progenitor cells into an organ suffering from an injury in a patient, and/or 3) pre-treatment of a patient subject to stem cell and/or progenitor cell therapy involving transplantation of stem cells and/or progenitor cells into said patient.

Yet another aspect of the embodiments relates to a method of inducing proliferation and/or differentiation in vitro of cells selected from a group consisting of stem cells and progenitor cells. The method comprises contacting the cells in vitro with an agonist of AT ₂ receptor.

A further aspect of the embodiments relates to a method of increasing viability of cells selected from a group consisting of stem cells and progenitor cells in in vitro cell cultures. The method comprises contacting the cells in vitro with an agonist of AT ₂ receptor.

Still another aspect of the embodiments relates to a method of reducing oxidative stress in cells selected from a group consisting of stem cells and progenitor cells in in vitro cell cultures. The method comprises contacting the cells in vitro with an agonist of AT ₂ receptor.

Further aspects of the embodiments include a method for inducing proliferation and/or differentiation of cells selected from a group consisting of stem cells and progenitor cells in a patient. The method comprises administering an agonist of AT ₂ receptor to the patient to induce proliferation and/or differentiation of the cells in the patient.

Another aspect of the embodiments is a method for inducing mobilization of cells selected from a group consisting of stem cells and progenitor cells into an organ suffering from an injury. The method comprises administering an agonist of AT ₂ receptor to a patient to induce mobilization of the cells into the organ in the patient.

Yet another aspect of the embodiments relates toa method for pre-treatment of a patient subject to subject to stem cell and/or progenitor cell therapy. The method comprises administering an agonist of AT ₂ receptor to the patient. The method also comprises transplanting stem cells and/or progenitor cells into the patient.

In one embodiment of the present invention, there is provided a method of adjuvant administration, which method comprises administration of a therapeutically effective amount of a compound of the invention (or a pharmaceutically acceptable salt, solvate or prodrug thereof) to a person for stem cell and progenitor cell therapy.

In another embodiment of the invention, compounds of the invention (e.g. AT ₂ receptor agonists or compounds that stimulate AT ₂ receptors), in particular non-peptide selective agonists of that sub-receptor or a pharmaceutically acceptable salt, solvate or prodrug thereof, may also be used in the manufacture of a medicament for adjuvant administration or for pre- treatment purposes in stem cell and progenitor cell therapy. In some embodiments, the compound can be N-butyloxycarbonyl-3-(4-imidazol-l-ylmethylphenyl)-5-z50- butylthiophene-2-sulfonamide (C21), or a pharmaceutically acceptable salt, solvate or prodrug thereof, which may be used in the manufacture of a medicament for adjuvant administration or for pre-treatment purposes in stem cell and progenitor cell therapy.

According to a further embodiment of the invention, there is provided a method for adjuvant administration in stem cell and progenitor cell therapy, which method comprises administering a compound of the invention (e.g. an AT ₂ receptor agonist or other compound that stimulates an AT ₂ receptor, or a pharmaceutically acceptable salt, solvate or prodrug thereof) to a patient in need of such treatment. In some embodiments, a method for adjuvant administration in stem cell and progenitor cell therapy, comprises administering the compound N-butyloxycarbonyl-3-(4-imidazol- 1 -ylmethylphenyl)-5-zso-butylthiophene-2- sulfonamide (C21), or a pharmaceutically acceptable salt, solvate or prodrug thereof, to a patient in need of such treatment.

Adjuvant administration or pre-treatment with a specific AT ₂ receptor agonist, in particular a non-peptide selective agonists of that sub-receptor, will provide a beneficial environment for stem cells and progenitor cells, resembling the fetal state with marked AT ₂ receptor activation, in which such cells are better programmed to differentiate into functioning organ specific cells.

Other embodiments and advantages will be more fully apparent from the following disclosure.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings are provided to illustrate various aspects of the present inventive concept and are not intended to limit the scope of the present invention unless specified herein.

Fig. 1 presents the structure of Compound 21 or, in short, C21, N-butyloxycarbonyl-3-(4- imidazo 1- 1 -ylmethylphenyl)-5 -zso-butylthiophene-2-sulfonamide.

Fig. 2 illustrates ffects of AT ₂ R activation on cell proliferation and oxidative stress in human PPCs. (A) PPC viability in response to series of STZ doses. (B) C21 effects on PPC viability. (C) C21 pre-conditioning rescues cell proliferative activity after STZ exposure. (D- E) DHE assay of superoxide levels. Scale bar = 50 μιη. (F) SOD1 mRNA expression in PPCs. All data are expressed as means ± SEM. N = 6 per group. *p < 0.05, **p < 0.01, ***p < 0.001 vs. Control, ^# p < 0.05, ^## p < 0.01, ^### p < 0.001 vs. STZ, ^δ ρ < 0.05 vs. STZ+C21.

DETAILED DESCRIPTION

The foregoing and other aspects of the present invention will now be described in more detail with respect to the description and methodologies provided herein. It should be appreciated that the invention may be embodied in different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art.

The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

The skilled person will understand that terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. Unless otherwise defined, all terms, including technical and scientific terms used in the description, have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

As used in the description of the embodiments of the invention, the singular forms "a", "an" and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise. Thus, such references may be replaced with a reference to "one or more" (e.g. one) of the relevant component or integer. As used herein, all references to "one or more" of a particular component or integer will be understood to refer to from one to a plurality (e.g. two, three or four) of such components or integers. It will be understood that references to "one or more" of a particular component or integer will include a particular reference to one such integer. Also, as used herein, "and/or" refers to and encompasses any and all possible combinations of one or more of the associated listed items. Furthermore, the term "about," as used herein when referring to a measurable value such as an amount of a compound, dose, time, temperature, and the like, refers to variations of 20%, 10%>, 5%, 1%, 0.5%, or even 0.1% of the specified amount. When a range is employed (e.g. a range from x to y) it is it meant that the measurable value is a range from about x to about y, or any range or value therein including x and y, such as about xi to about yi, etc. It will be further understood that the terms "comprises" and/or "comprising," when used in this specification, specify the presence of stated features, integers, steps, operations, elements, components and/or groups, but do not preclude the presence or addition of one or more other features, integers, steps, operations, elements, components, and/or groups thereof. Unless otherwise defined, all terms, including technical and scientific terms used in the description, have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

"Effective amount" as used herein refers to an amount of a compound, composition and/or formulation of the invention that is sufficient to produce a desired effect, which can be a therapeutic and/or beneficial effect. The effective amount will vary with the age, general condition of the subject, the severity of the condition being treated, the particular agent administered, the duration of the treatment, the nature of any concurrent treatment, the pharmaceutically acceptable carrier used, and like factors within the knowledge and expertise of those skilled in the art. As appropriate, an "effective amount" in any individual case can be determined by one skilled in the art by reference to the pertinent texts and literature and/or by using routine experimentation.

By the term "treat," "treating," or "treatment of (and grammatical variations thereof) it is meant that the severity of the subject's condition is reduced, at least partially improved or ameliorated and/or that some alleviation, mitigation or decrease in at least one clinical symptom is achieved and/or there is a delay in the progression of the disease or disorder.

A "therapeutically effective" amount as used herein is an amount that is sufficient to treat (as defined herein) the subject. Those skilled in the art will appreciate that the therapeutic effects need not be complete or curative, as long as some benefit is provided to the subject.

The term "therapeutically effective" as used herein in reference to an amount or dose refers to an amount of a compound, composition and/or formulation of the invention that is sufficient to produce a desired effect, which can be a therapeutic and/or beneficial effect. Such an effective amount will vary with the age, general condition of the subject, the severity of the condition being treated, the particular agent administered, the duration of the treatment, the nature of any concurrent treatment, the pharmaceutically acceptable carrier used, and like factors within the knowledge and expertise of those skilled in the art. As appropriate, an effective amount in any individual case can be determined by one skilled in the art by reference to the pertinent texts and literature and/or by using routine experimentation.

A "subject in need" of the methods of the invention can be a subject undergoing stem cell or progenitor cell therapy.

As used herein the term "concomitant administration" or "combination administration" of a compound, therapeutic agent or known drug with a compound of the present invention means administration of a known medication or drug and, in addition, the one or more compounds of the invention at such time that both the known drug and the compound will have a therapeutic effect. In some cases this therapeutic effect will be synergistic. Such concomitant administration can involve concurrent (i.e. at the same time), prior, or subsequent administration of the known drug with respect to the administration of a compound of the present invention. A person skilled in the art, would have no difficulty determining the appropriate timing, sequence and dosages of administration for particular drugs and compounds of the present invention.

In addition, in some embodiments, the compounds of this invention will be used, either alone or in combination with each other or in combination with one or more other therapeutic medications as described above, or their pharmaceutically acceptable salts, solvates or prodrugs, for manufacturing a medicament for the purpose of providing adjuvant

administration for stem cell and progenitor cell therapy.

Pharmaceutically acceptable salts include, but are not limited to, acid addition salts and base addition salts. Such salts may be formed by conventional means, for example by reaction of a free acid or a free base form of a compound of the invention with one or more equivalents of an appropriate acid or base, optionally in a solvent, or in a medium in which the salt is insoluble, followed by removal of said solvent, or said medium, using standard techniques (e.g. in vacuo or by freeze-drying). Salts may also be prepared by exchanging a counter-ion of a compound of the invention in the form of a salt with another counter-ion, for example, using a suitable ion exchange resin. For the avoidance of doubt, other pharmaceutically acceptable derivatives of compounds of the invention are included within the scope of the invention (e.g. solvates, prodrugs etc.).

As used herein, a "prodrug" is a composition that undergoes an in vivo modification when administered to a subject, wherein the product of the in vivo modification is a therapeutically effective compound. Prodrugs of compounds may be prepared by, for example, preparing a given compound as an ester. Thus, for example, an esterified form of the compound may be administered to a subject and may be de-esterified in vivo thereby releasing a therapeutically effective compound. Alternatively, some compounds may be prepared as prodrugs by adding short polypeptides (e.g. 1-6 amino acids) to the compound. Such prodrugs when administered to a subject may be cleaved (by, e.g. trypsin or other peptidases/proteases) thereby releasing a therapeutically effective compound. Formation of prodrugs is not limited by the specific examples described herein. Other ways of preparing therapeutically effective compounds as prodrugs are known.

Compounds of the invention may exhibit tautomerism. All tautomeric forms and mixtures thereof are included within the scope of the invention.

Compounds of the invention also contain one or more asymmetric carbon atoms and may therefore exhibit optical and/or diastereoisomerism. Diastereoisomers may be separated using conventional techniques, e.g. chromatography or fractional crystallization. The various stereoisomers may be isolated by separation of a racemic or other mixture of the compounds using conventional, e.g. fractional crystallization or HPLC, techniques. Alternatively, the desired optical isomers may be made by reaction of the appropriate optically active starting materials under conditions which will not cause racemisation or epimerisation, or by derivatisation, for example with a homochiral acid followed by separation of the

diastereomeric derivatives by conventional means (e.g. HPLC, chromatography over silica). All stereoisomers are included within the scope of the invention.

All patents, patent applications and publications referred to herein are incorporated by reference in their entirety. In the event of conflicting terminology, the present specification is controlling. Further, the embodiments described in one aspect of the present invention are not limited to the aspect described. The embodiments may also be applied to a different aspect of the invention as long as the embodiments do not prevent these aspects of the invention from operating for their intended purpose.

Accumulating evidence show that AT ₂ receptors in man are involved in the healing process of organ damage. As disclosed in the present invention, stimulation of the AT ₂ receptor with a specific agonist, exemplified but not limited to C21, is able to amplify these healing mechanisms in a number of tissues with the provision of growth and differentiation of local undifferentiated cells.

Accordingly, in a first aspect of the invention, there is provided an AT ₂ receptor agonist (or a compound that stimulates AT ₂ receptors), in particular a non-peptide AT ₂ receptor agonist or a pharmaceutically acceptable salt (preferably an HC1 salt of a compound of the invention), solvate or prodrug thereof, for use in the adjuvant treatment or pre-treatment for stem cell and progenitor cell therapy. Such AT ₂ receptor agonists and/or compounds that stimulate AT ₂ receptors may be referred to herein as the "compounds of the invention". In some embodiments, a compound of the invention can include, but is not limited to, N- butyloxycarbonyl-3-(4-imidazol- 1 -ylmethylphenyl)-5-z50-butylthiophene-2-sulfonamide (C21).

Thus, a compound of the invention includes AT ₂ receptor agonists that fully and those that partially activate the AT ₂ receptor and those compounds that can stimulate or activate the AT ₂ receptor. In some embodiments, an AT ₂ receptor agonist may be defined to include any compound that can stimulate or activate the AT ₂ receptor. In some embodiments, the compound of the invention is an AT ₂ receptor specific agonist that binds selectively to the AT ₂ receptor. In some embodiments, the compound of the invention is a non-peptide AT ₂ receptor specific agonist that binds selectively to the AT ₂ receptor.

Non-limiting examples of compounds of the invention include N-butyloxycarbonyl-3-

(4-imidazol-l-ylmethylphenyl)-5-z50-butylthiophene-2-sulf onamide (C21) or a

pharmaceutically acceptable salt, solvate or prodrug thereof.

Non-limiting examples of compounds of the invention that bind selectively to the AT ₂ receptor include N-butyloxycarbonyl-3-(4-imidazol- 1 -ylmethylphenyl)-5-zso-butylthiophene- 2-sulfonamide (C21).

According to a another aspect of the invention, there is provided the use of a compound of the invention, for example, the compound N-butyloxycarbonyl-3-(4-imidazo 1-1 - ylmethylphenyl)-5-z50-butylthiophene-2-sulfonamide (C21) or a pharmaceutically acceptable salt (preferably an HC1 salt of a compound of the invention), solvate or prodrug thereof, for adjuvant administration or for pre-treatment purposes in stem cell and progenitor cell therapy.

In another aspect of the invention, the compounds of the invention (e.g. AT ₂ receptor agonists, and other compounds that stimulate AT ₂ receptors), or a pharmaceutically acceptable salt (e.g. an HC1 salt of the compound of the invention), solvates or prodrug thereof, may also be used in the manufacture of a medicament for adjuvant administration or for pre-treatment purposes in stem cell and progenitor cell therapy.

According to a another aspect of the invention, there is provided a method for adjuvant administration in stem cell and progenitor cell therapy, which method comprises

administering a compound of the invention or a pharmaceutically acceptable salt, solvate or prodrug thereof, to a patient in need of such treatment. In some embodiments, the compound of the invention can be N-butyloxycarbonyl-3-(4-imidazol- 1 -ylmethylphenyl)-5 -z ^' so- butylthiophene-2-sulfonamide (C21), or a pharmaceutically acceptable salt (preferably an HC1 salt of the compound of the invention), solvate or prodrug thereof.

The compound N-butyloxycarbonyl-3-(4-imidazol- 1 -ylmethylphenyl)-5 -z ^' so- butylthiophene-2-sulfonamide (C21) with the structure provided in Fig. 1, may be made in accordance with techniques well known to those skilled in the art, for example as described in international patent application WO 02/096883, all of its content hereby incorporated by reference, in particular with regard to the production of C21 and more in particular as shown in Example 2 on pages 34 to 38 in WO 02/096883.

The compounds of the invention are useful because they possess pharmacological activity. In particular, the compounds of the invention are agonists of Ang II receptor, more particularly, they are agonists of the AT ₂ receptor, and, especially, are selective agonists of that sub-receptor, in particular non-peptide selective agonists of that sub-receptor. Compounds of the invention have the advantage that they bind selectively to, and exhibit agonist activity at, the AT ₂ receptor. By compounds that "bind selectively" to the AT ₂ receptor, we include that the affinity ratio for the relevant compound (AT ₂ :ATi) is at least 50: 1, at least 100: 1, preferably at least 1000: 1, more preferably at least 10000: 1, and even more preferably at least 25000: 1.

The present inventors have found that the compounds of the invention (e.g. AT ₂ receptor agonists) are useful for adjuvant administration or for pre-treatment purposes in stem cell and progenitor cell therapy. According to a further aspect of the present invention, there is provided a method for adjuvant administration in stem cell and progenitor cell therapy, which method comprises administration of a therapeutically effective amount of a compound of the invention or a pharmaceutically acceptable salt thereof (e.g, an HC1 salt of a compound of the invention), solvate or prodrug thereof to a subject in need of such treatment.

According to a further aspect of the present invention, there is provided a pre-treatment method for adjuvant administration in stem cell and progenitor cell therapy, which pre- treatment method comprises administration of a therapeutically effective amount of a compound of the invention or a pharmaceutically acceptable salt thereof (e.g, an HC1 salt of a compound of the invention), solvate or prodrug thereof to a subject in need of such treatment.

Beneficial effects of C21 can be observed on human pancreatic progenitor cells (PPCs), which serve as a stem cell model to study the property of cell differentiation and regeneration. The inventors demonstrate that AT ₂ R activation induced protection of human PPCs from STZ (streptozotocin) via increased cell proliferation and reduction of oxidative stress.

According to a further aspect of the present invention, there is provided a pre-treatment method for increased cell proliferation and reduction of oxidative stress, which pre-treatment method comprises administration of a therapeutically effective amount of a compound of the invention or a pharmaceutically acceptable salt thereof (e.g, an HC1 salt of a compound of the invention), solvate or prodrug thereof to a subject in need of such treatment.

STZ evoked a dose-dependent reduction in the viability of PCCs, as shown by MTT assay data, compared to Control cells (Fig. 2A), whereas C21 pre-conditioning alone did not affect cell viability (Fig. 2B). However, 1 μΜ and 10 μΜ C21 pre-conditioning resulted in improved cell viability (by 20-30%) in STZ-exposed cells, versus STZ only cells. This protective effect of C21 was abolished by AT ₂ R antagonism with 10 μΜ PD123319 (Fig. 2C). According to a further aspect of the present invention, there is provided a pre-treatment or pre-conditioning method for increased cell viability by inducing protective effect, which pre-treatment method comprises administration of a therapeutically effective amount of a compound of the invention or a pharmaceutically acceptable salt thereof (e.g. an HC1 salt of a compound of the invention), solvate or prodrug thereof to a subject in need of such treatment or to cells in vitro.

Oxidative stress, as reflected by DHE intensity, in STZ-treated cells was markedly reduced in the cells pretreated with C21 before STZ exposure (Fig. 2D), and the C21 effect was again abolished by 10 μΜ PD123319 (Fig. 2D and 2E). Similarly, SODl mRNA levels were significantly down-regulated in STZ-treated cells, and this down-regulation was reversed by pretreatment with C21 (Fig. 2F).

According to a further aspect of the present invention, there is provided a pre-treatment method for reversal of down-regulation of SODl mRNA levels, which pre-treatment method comprises administration of a therapeutically effective amount of a compound of the invention or a pharmaceutically acceptable salt thereof (e.g. an HC1 salt of a compound of the invention), solvate or prodrug thereof to a subject in need of such treatment.

The DHE staining results and the MTT assay results were consistent with the possibility that the protective mechanism of C21 's effects may involve proliferative and anti-oxidative pathways.

An aspect of the embodiments relates to an agonist of AT ₂ receptor for use in inducing proliferation and/or differentiation of cells selected from a group consisting of stem cells and progenitor cells.

The stem cells could be hematopoietic stem cells and/or mesenchymal stem cells. Correspondingly, the progenitor cells could be, for instance, epithelial stem cells, intermediate progenitor cells, bone marrow stromal cells, pancreatic progenitor cells, angioblasts, endothelial progenitor cells, blast cells and/or satellite cells.

In an embodiment, the agonist is for use in inducing proliferation and/or differentiation of the cells in a patient subject to stem cell and/or progenitor cell therapy.

In an embodiment, the agonist is for use in inducing differentiation of the cells into organ specific cells.

In a particular embodiment, the agonist is for use in inducing differentiation of the cells into organ specific cells to improve healing of a patient suffering from an injury in the organ.

Thus, the AT ₂ receptor agonist of the embodiments is capable of inducing differentiation of stem cells and progenitor cells into differentiated cells in the form of so called organ specific cells. This means that the stem and/or progenitor cells are differentiated into cell types generally present in the relevant organ or tissue and exerting a function in the relevant organ or tissue that is the same or at least similar to the function of the specific cells present in and/or constituting the relevant organ or tissue.

The induced cell differentiation implies that the stem and/or progenitor cells undergoes a cell change from one cell type (stem cell type or progenitor cell type) to another cell type that is generally a more specialized type as compared to stem cells and progenitor cells. Cell differentiation may involve changes in the cell's size, shape, membrane potential, metabolic activity, responsiveness to signals, etc. Such changes are largely due to controlled modifications in gene expressions as the cells differentiate from stem or progenitor cells into the organ specific cells. Accordingly, following differentiation, the differentiated cells have characteristics and gene expression profiles similar to at least one cell type that is present in the relevant organ or tissue.

In an embodiment, the agonist is for use in reducing oxidative stress in the cells.

Another aspect of the embodiments relates to an agonist of AT ₂ receptor for use in inducing mobilization of cells selected from a group consisting of stem cells and progenitor cells into an organ suffering from an injury in a patient.

In an embodiment, the agonist is for use in inducing mobilization of the cells into the organ and inducing differentiation of the cells into organ specific cells in the organ.

For instance, the AT ₂ receptor agonist could be administered to a patient following a myocardial infarction (MI). In such a case, the AT ₂ receptor agonist will induce mobilization of stem cells and/or progenitor cells and differentiation thereof into myocardial cells that may repair the injury caused by the MI in the patient's heart.

A further aspect of the embodiments relates to an agonist of AT ₂ receptor for use in pre- treatment of a patient subject to stem cell and/or progenitor cell therapy involving transplantation of stem cells and/or progenitor cells into the patient.

In this embodiment, the patient is subject to a so called pre-treatment or pre-conditioning in terms of administration of the AT ₂ receptor agonist preferably at least prior to transplantation or injection of stem cells and/or progenitor cells into the patient as a part of stem cell and/or progenitor cell therapy. Thus, the AT ₂ receptor agonist administration may take place prior to cell transplantation in order to provide a favorable environment within the patient body once the stem cells and/or progenitor cells are transplanted. The administration of the AT ₂ receptor may also, or alternatively, take place simultaneously as and/or following transplantation of the stem cells and/or progenitor cells. Hence, the embodiments include administering the AT ₂ receptor agonist prior to cell transplantation, simultaneously as cell transplantation, following cell transplantation, prior to cell transplantation and simultaneously as cell transplantation, prior to cell transplantation and following cell transplantation, simultaneously as cell transplantation and following cell transplantation or prior to cell transplantation, simultaneously as cell transplantation and following cell transplantation.

Simultaneous administration of the AT ₂ receptor agonist and cell transplantation can be performed according to various embodiments. For instance, the AT ₂ receptor agonist may be administered at the same site or another site in the patient body as the site for cell transplantation. If administered at the same site, the AT ₂ receptor agonist may be administered separate from the stem cells and/or progenitor cells and/or could be added to the cell solution comprising the stem cells and/or progenitor cells and a vehicle.

In an embodiment, the agonist is a non-peptide agonist that binds selectively to the AT ₂ receptor. In a particular embodiment, the agonist is N-butyloxycarbonyl-3-(4-imidazol-l- ylmethylphenyl)-5-z50-butylthiophene-2-sulfonamide or a pharmaceutically acceptable salt, solvate or prodrug thereof.

The compounds of the invention will normally be administered orally, intravenously, subcutaneously, buccally, rectally, dermally, nasally, tracheally, bronchially, by any other parenteral route or via inhalation, or combinations thereof, in a pharmaceutically acceptable dosage form. Additional methods of administration include but are not limited to intraarterial, intramuscular, intraperitoneal, intraportal, intradermal, epidural, and/or intrathecal

administration or combinations thereof.

The compounds of the invention may be administered alone, but are preferably administered by way of known pharmaceutical formulations, including tablets, capsules or elixirs for oral administration, suppositories for rectal administration, sterile solutions or suspensions for parenteral or intramuscular administration, and the like.

Such formulations may be prepared in accordance with standard and/or accepted pharmaceutical practice.

According to a further aspect of the invention there is thus provided a pharmaceutical formulation comprising a compound of the invention, in admixture with a pharmaceutically acceptable adjuvant, diluent or carrier, for adjuvant administration in stem cell and progenitor cell therapy.

Compounds of the invention may also be administered in combination with other AT ₂ agonists that are known in the art, as well as in combination with ATi receptor antagonists that are known in the art, such as losartan, and/or in combination with an inhibitor of angiotensin converting enzyme (ACE). Such combinations may therefore be useful for adjuvant administration in stem cell and progenitor cell therapy.

According to a further aspect of the invention, there is provided a combination product comprising:

• (A) an AT ₂ receptor agonist and/or a compound that stimulates AT ₂ receptors, and

• (B) an ATi receptor antagonist, and/or an ACE inhibitor,

wherein each of components (A) and (B) is formulated in admixture with a pharmaceutically- acceptable adjuvant, diluent or carrier, for adjuvant administration in stem cell and progenitor cell therapy.

Non-limiting but illustrative examples of ATi receptor antagonists that can be used according to the embodiments include losartan, irbesartan, olmesartan, candesartan, valsartan, fimasartan, telmisartan, azilsartan, eprosartan, milfasartan, pomisartan, pratosartan, ripiasartan, saprisartan, tasosartan and combinations thereof.

Non- limiting but illustrative examples of ACE inhibitors that can be used according to the embodiments include sulfhydryl-containing agents, such as captopril and zofenopril; dicarboxylate-containing agents, such as enalapril, ramipril, quinapril, perindopril, lisinopril, benazepril, imidapril, trandolapril and cilazapril; phosphonate-containing agents, such as fosinopril; casokinins; lactokinins; lactotripeptides, such as Val-Pro-Pro and Ile-Pro-Pro, or combinations thereof. Further ACE inhibitors that can be used according to the embodiments include moexipril, spirapril, temocapril, alacepril, ceronapril, delepril, moveltipril and combinations thereof.

Such combination products provide for the administration of an AT ₂ receptor agonist and/or a compound that stimulates an AT ₂ receptor (as defined herein), in particular non- peptide selective agonists of that sub-receptor, in conjunction with an ATi receptor antagonist and/or an ACE inhibitor, and may thus be presented either as separate formulations, wherein at least one of those formulations comprises an AT ₂ receptor agonist or a compound that stimulates an AT ₂ receptor (as defined herein, e.g. a compound of the invention, or a pharmaceutically acceptable salt, solvate or prodrug thereof), and at least one formulation comprises an ATi receptor antagonist and/or an ACE inhibitor, or may be presented (i.e. formulated) as a combined preparation (i.e. presented as a single formulation including an AT ₂ receptor agonist and/or a compound that stimulates an AT ₂ receptor together with either an ATi receptor antagonist or an ACE inhibitor).

Thus, there is further provided: (1) a pharmaceutical formulation comprising an AT ₂ receptor agonist and/or a compound that stimulates an AT ₂ receptor (e.g. a compound of the invention, or a

pharmaceutically acceptable salt, solvate or prodrug thereof) and an ATi receptor antagonist or an ACE inhibitor in admixture with a pharmaceutically-acceptable adjuvant, diluent and/or carrier, for adjuvant administration in stem cell and progenitor cell therapy; and

(2) a kit of parts comprising components:

• (a) a pharmaceutical formulation comprising an AT ₂ receptor agonist and/or a compound that stimulates an AT ₂ receptor (e.g. a compound of the invention, or a pharmaceutically acceptable salt, solvate or prodrug thereof), in admixture with a pharmaceutically-acceptable adjuvant, diluent and/or carrier; and

• (b) a pharmaceutical formulation including an ATi receptor antagonist, or an ACE inhibitor, in admixture with a pharmaceutically-acceptable adjuvant, diluent and/or carrier,

which components (a) and (b) are each provided in a form that is suitable for administration in conjunction with the other, for adjuvant administration in stem cell and progenitor cell therapy.

An aspect of the embodiments relates to a composition comprising an agonist of AT ₂ receptor and an antagonist of ATi receptor and/or an ACE inhibitor for use in inducing proliferation and/or differentiation of cells selected from a group consisting of stem cells and progenitor cells.

Another aspect of the embodiments relates to a composition comprising an agonist of AT ₂ receptor and an antagonist of ATi receptor and/or an ACE inhibitor, for use in inducing mobilization of cells selected from a group consisting of stem cells and progenitor cells into an organ suffering from an injury in a patient.

A further aspect of the embodiments relates to a composition comprising an agonist of

AT ₂ , receptor and an antagonist of ATi receptor and/or an ACE inhibitor for use in pre- treatment of a patient subject to stem cell and/or progenitor cell therapy involving transplantation of stem cells and/or progenitor cells into the patient.

Further aspects of the embodiments includes a method of inducing proliferation and/or differentiation in vitro of cells selected from a group consisting of stem cells and progenitor cells. The method comprises contacting the cells in vitro with an agonist of AT ₂ receptor.

Another aspect relates to a method of increasing viability of cells selected from a group consisting of stem cells and progenitor cells in in vitro cell cultures. The method comprises contacting the cells in vitro with an agonist of AT ₂ receptor. A further aspect of the embodiments includes a method of reducing oxidative stress in cells selected from a group consisting of stem cells and progenitor cells in in vitro cell cultures. The method comprises contacting the cells in vitro with an agonist of AT ₂ receptor.

In an embodiment, contacting the cells comprises contacting the cells in vitro with a composition comprising the agonist of AT ₂ , receptor and an antagonist of ATi receptor and/or ACE inhibitor.

The treatment or conditioning of stem cells and/or progenitor cells according to the above described aspects and embodiments can be used in connection with stem cell and/or progenitor cell therapy. Thus, stem cells and/or progenitor cells that have been pre-treated or pre- conditioned with the AT ₂ receptor agonist or the AT ₂ receptor agonist comprising composition could be transplanted into a patient. In this case, the patient may additionally have been pre- treated with the AT ₂ receptor agonist or composition and/or is treated with the agonist or composition simultaneously and/or following the transplantation of the treated stem cells and/or progenitor cells.

The source of the stem cells and/or progenitor cells treated by the AT ₂ receptor agonist or composition could be the same patient that will subsequently receive the treated stem cells and/or progenitor cells, another patient or indeed any stem cell and/or progenitor cell source.

For instance, progenitor cells could be obtained from the skin of a patient, treated with the AT ₂ receptor agonist or composition and then be used to repair other organs in the patient or in another patient. The pre-conditioning of the progenitor cells with the AT ₂ receptor agonist increases the capability of the progenitor cells to differentiate into organ specific cells and improves the survival rate and viability of the progenitor cells.

In this context, the cells treated with the AT ₂ receptor agonist or composition could be individual cells, clusters of cells or indeed tissues or organs, possible comprising a plurality of cells of possibly varying cell types. In an embodiment, the tissue or organ to which stem cells and/or progenitor cells should be mobilized and differentiated may be treated with, i.e. contacted with, the AT ₂ receptor agonist. For instance, the tissue or organ could be treated ex vivo and then transplanted into a recipient patient. The AT ₂ receptor agonist pre-conditioning of the transplanted tissue or organ provides a favorable environment for patient's stem cells and/or progenitor cells, or transplanted stem cells and/or progenitor cells. Accordingly, the stem cells and/or progenitor cells can differentiate into specialized organ specific cells at the transplanted tissue or organ and may, for instance, protect the transplanted tissue or organ during the initial phases of engraftment. A further aspect of the embodiments relates to a method for inducing proliferation and/or differentiation of cells selected from a group consisting of stem cells and progenitor cells in a patient. The method comprises administering an agonist of AT ₂ receptor to the patient to induce proliferation and/or differentiation of the cells in the patient.

Another aspect of the embodiments relates to a method for inducing mobilization of cells selected from a group consisting of stem cells and progenitor cells into an organ suffering from an injury. The method comprises administering an agonist of AT ₂ receptor to a patient to induce mobilization of the cells into the organ in the patient.

Yet another aspect of the embodiments relates to a method for pre-treatment of a patient subject to subject to stem cell and/or progenitor cell therapy. The method comprises administering an agonist of AT ₂ receptor to the patient. The method also comprises transplanting stem cells and/or progenitor cells into the patient.

In an embodiment, the method also comprises contacting the stem cells and/or progenitor cells in vitro with the agonist of AT ₂ receptor.

In an embodiment, administering the agonist comprises administering a composition comprising the agonist of AT ₂ receptor and an ATi receptor antagonist and/or ACE inhibitor to the patient.

Depending upon the patient to be treated and the route of administration, the compounds of the invention may be administered at varying doses. Although doses will vary from patient to patient, suitable daily doses are in the range of about 1 to 1000 mg (e.g. 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000 mg, and the like, or any range or value therein) per patient, administered in single or multiple doses. More preferred daily doses are in the range 2.5 to 250 mg (e.g. 2.5, 3, 3.5, 4. 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250 mg and the like or any range or value therein) per patient.

Individual doses of compounds of the invention may be in the range 1 to 100 mg (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, and the like, or any range or values therein). Advantageously, compounds of the present invention may be administered in single doses, e.g. once daily or more seldom, or in a total daily dosage administered in divided doses of two, three or four times daily.

In any event, the physician, or the skilled person, will be able to determine the actual dosage which will be most suitable for an individual patient, which is likely to vary with the condition that is to be treated, as well as the age, weight, sex and response of the particular patient to be treated. The above-mentioned dosages are exemplary of the average case; there can, of course, be individual instances where higher or lower dosage ranges are merited, and such are within the scope of this invention.

The compounds of the invention may also have the advantage that they may be more efficacious than, be less toxic than, be longer acting than, be more potent than, produce fewer side effects than, be more easily absorbed than, and/or have a better pharmacokinetic profile (e.g. higher oral bioavailability and/or lower clearance) than, and/or have other useful pharmacological, physical, or chemical properties than compounds known in the prior art. Such effects may be evaluated clinically, objectively and/or subjectively by a health care professional, a treatment subject or an observer.

Subjects or patients suitable to be treated with formulations of the present invention include, but are not limited to, mammalian subjects. In some embodiments, the subject can be a human subject.

Accordingly, in some embodiments, a method for adjuvant administration in stem cell and progenitor cell therapy in a subject in need thereof is provided, comprising: administering to said subject a therapeutically effective amount of an angiotensin II (AT ₂ ) receptor agonist and/or a pharmaceutically acceptable salt, solvate, solvate of a pharmaceutically acceptable salt, or prodrug thereof.

Accordingly, in some embodiments, a pre-treatment method for adjuvant administration in stem cell and progenitor cell therapy in a subject in need thereof is provided, comprising: pre-treatment and administering to said subject a therapeutically effective amount of an angiotensin II (AT ₂ ) receptor agonist and/or a pharmaceutically acceptable salt, solvate, solvate of a pharmaceutically acceptable salt, or prodrug thereof.

In some embodiments, the AT ₂ receptor agonist is an AT ₂ specific agonist, in particular a non-peptide selective agonists of that sub-receptor. In some embodiments, the AT ₂ receptor agonist is N-butyloxycarbonyl-3-(4-imidazol- 1 -ylmethylphenyl)-5-zso-butylthiophene-2- sulfonamide (C21) or a pharmaceutically acceptable salt, solvate, solvate of pharmaceutically acceptable salt, or prodrug thereof. In some embodiments, the AT ₂ receptor agonist is the compound depicted in Fig. 1.

In some embodiments, the method comprises administering to said subject a therapeutically effective amount of an ATi receptor antagonist and/or an angiotensin converting enzyme (ACE) inhibitor. In some embodiments, the AT ₂ receptor agonist is provided in the same composition or pharmaceutical formulation as the ATi receptor antagonist and/or the ACE inhibitor. In some embodiments, AT ₂ receptor agonist is provided in a different formulation from the ATi receptor antagonist and/or the ACE inhibitor and the separate formulations are administered to the subject simultaneously, sequentially or separately. In some embodiments, the composition or pharmaceutical formulation comprises a pharmaceutically-acceptable adjuvant, diluent and/or carrier. In some embodiments, administering comprises oral, intravenous, subcutaneous, buccal, rectal, dermal, nasal, tracheal, bronchial, inhalation, intraarterial, intramuscular, intraperitoneal, intraportal, intradermal, epidural, and/or intrathecal administration or combinations thereof.

In some embodiments, the present invention provides a kit for adjuvant administration in stem cell and progenitor cell therapy comprising a pharmaceutical formulation comprising AT ₂ receptor agonist. In some embodiments, the present invention provides a kit for pre- treatment and adjuvant administration in stem cell and progenitor cell therapy comprising a pharmaceutical formulation comprising AT ₂ receptor agonist. In some embodiments, the kit further comprises a pharmaceutical formulation comprising an ATi receptor antagonist and/or an ACE inhibitor. In some embodiments, the kit further comprises instructions for adjuvant administration or pre-treatment with an effective amount of the pharmaceutical formulation. In some embodiments, the kit further comprises an AT ₂ receptor agonist that is the AT ₂ receptor agonist depicted in Fig.1.

In one embodiment of the present invention, there is provided a method of adjuvant administration, which method comprises administration of a therapeutically effective amount of a compound of the invention (or a pharmaceutically acceptable salt, solvate or prodrug thereof) to a person for stem cell and progenitor cell therapy. In one embodiment of the present invention, there is provided a pre-treatment method of adjuvant administration, which pre-treatment method comprises administration of a therapeutically effective amount of a compound of the invention (or a pharmaceutically acceptable salt, solvate or prodrug thereof) to a person for stem cell and progenitor cell therapy.

In another embodiment of the invention, compounds of the invention (e.g. AT ₂ receptor agonists or compounds that stimulate AT ₂ receptors), or a pharmaceutically acceptable salt, solvate or prodrug thereof, may also be used in the manufacture of a medicament for adjuvant administration in stem cell and progenitor cell therapy. In another embodiment of the invention, compounds of the invention (e.g. AT ₂ receptor agonists or compounds that stimulate AT ₂ receptors), or a pharmaceutically acceptable salt, solvate or prodrug thereof, may also be used in the manufacture of a medicament for pre-treatment and adjuvant administration in stem cell and progenitor cell therapy. In some embodiments, the compound can be N-butyloxycarbonyl-3-(4-imidazol- 1 -ylmethylphenyl)-5-zso-butylthiophene-2- sulfonamide (C21), or a pharmaceutically acceptable salt, solvate or prodrug thereof, which may be used in the manufacture of a medicament for adjuvant administration or pre-treatment in stem cell and progenitor cell therapy.

According to a further embodiment of the invention, there is provided a method for adjuvant administration or for pre-treatment purposes in stem cell and progenitor cell therapy, which method comprises administering a compound of the invention (e.g. an AT ₂ receptor agonist or other compound that stimulates an AT ₂ receptor, or a pharmaceutically acceptable salt, solvate or prodrug thereof) to a patient in need of such treatment. In some embodiments, a method for adjuvant administration in stem cell and progenitor cell therapy, comprises administering the compound N-butyloxycarbonyl-3-(4-imidazol-l-ylmethylphenyl)-5-z50- butylthiophene-2-sulfonamide (C21), or a pharmaceutically acceptable salt, solvate or prodrug thereof, to a patient in need of such treatment.

In some embodiments an AT ₂ receptor agonist, in particular a non-peptide selective agonists of that sub-receptor, mobilizes stem cells from outside an organ, which then differentiate into functional organ specific tissue in the specific organ.

In some embodiments an AT ₂ receptor agonist, in particular a non-peptide selective agonists of that sub-receptor, stimulate progenitor cells to grow and differentiate into functional organ specific tissue.

In some embodiments an AT ₂ receptor agonist, in particular a non-peptide selective agonists of that sub-receptor, improves differentiation and growth of stem cells both in vitro and transplanted into the specific organs with improved functionality.

In some embodiments an AT ₂ receptor agonist, in particular a non-peptide selective agonists of that sub-receptor, enhance growth, proliferation, reduce oxidative stress and increase survival and cell viability of stem cells in cell cultures.

In some embodiments an AT ₂ receptor agonist, in particular a non-peptide selective agonists of that sub-receptor, enhance growth and differentiation of progenitor cells to organ specific cells.

In some embodiments an AT ₂ receptor agonist, in particular a non-peptide selective agonists of that sub-receptor, facilitate integration of transplanted stem cells into organs.

In some embodiments an AT ₂ receptor agonist, in particular a non-peptide selective agonists of that sub-receptor, facilitate a therapeutic for local reparation/transplantation of non-local undifferentiated cells. EXAMPLES

The STZ-injected neonatal rat model was employed because it has been well established for studies of β-cell regeneration by various groups. In this model, newborn rats sustain β-cell damage immediately after an STZ injection, and then show spontaneous remission with reduced risk of infection from other surgical operations (Thyssen et al, Endocrinology 2006; 147: 2346-2356, Wang et al, Diabetologia 1 §994; 37: 1088-1096).

Dihydroethidium (DHE) staining and analysis

Intracellular ROS levels were detected with DHE, a reduced form of the DNA dye ethidium bromide, as described previously (Wang et al, PLoS One 2015: eO 128216). Freshly dissected pancreatic rudiments were embedded in O.C.T compound and frozen immediately in liquid nitrogen for cryosectioning preparation. The slide-affixed sections were incubated in freshly prepared 10 μΜ DHE solution (Sigma- Aldrich, St. Louis, MO) for 30 min in a dark humidified chamber at 37 °C and then incubated with DAPI (Invitrogen) for 5 min before being cover-slipped with vectashield. The DHE solution was prepared by dissolving in

DMSO at 30 mM and then further diluting the solution to 10 μΜ DHE in DMSO. All digital images were acquired by a fluorescence microscope equipped with a DC200 digital camera and fluorescence intensity was quantified by Leica Qwin image analysis software (all from Leica Microsystems). Each group was represented by 9-12 images collected from three tissue blocks, each containing 6-8 embryos.

Fetal tissue procurement, processing, and PPC culture

Human fetal pancreata were provided by the Department of Obstetrics and Gynecology in the Prince of Wales Hospital, CUHK. Maternal consent and ethical approval were obtained from the Clinical Research Ethics Committee (CREC-2010.574) at our institution prior to the processing of specimens. The procedures for digestion of human fetal pancreas, and the subsequent isolation and culturing of PPCs were performed, as described previously with slight modifications (Leung et al, Stem Cells 2012; 30: 525-536). Briefly, each fetal pancreas was minced and digested in 3mg/ml collagenase P (Roche, Indianapolis, IN). Digested cells were re-suspended in RPMI 1640 media supplemented with 10% fetal bovine serum, 1 mM sodium pyruvate, 1% penicillin and streptomycin, 10 mM HEPES buffer (Invitrogen, Carlsbad, CA), and 71.5 μΜ β- mercaptoethanol (Sigma- Aldrich, St. Louis, MO) in 60-mm non-treated suspension culture dishes (Corning Incorporated, NY) and incubated at 37 °C in a 5% C0 ₂ /95% humidified incubator (Thermo Scientific, MA). PPC outgrowth from the nonadherent cell clusters was induced by supplementation of the medium with 20 ng/ml each of epidermal growth factor (Invitrogen) and basic fibroblast growth factor (Sigma- Aldrich). Monolayers of PPCs that migrated out from the cell clusters were harvested for subsequent cultures.

Assessment of PPC proliferation

Cells were plated 5,000 cells/well in 96-well plates with media containing C21 at one of three concentrations (0.1, 1, or 10 μΜ) for 2 d. In some experiments, the AT ₂ R antagonist PD 123 ,319 ( 10 μΜ; Sigma- Aldrich) was applied 10 min before the addition of C21 for 2 d. STZ (5 mM) was then added to the medium for 30 min. Cell proliferative activity and cell viability were assessed by 5-bromo-2'-deoxyuridine (BrdU) and MTT [3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assays. Data were calculated as percentages of initial control values for each treatment group (experimental value/initial control value xl00%), as described previously (Leung et al, Stem Cells 2012; 30: 525-536). The BrdU-incorporation assay (Roche Diagnostic, Mannheim, Germany) was performed according to the manufacturer's protocol. Briefly, cells grown in 96-well plates were labelled by exposure to BrdU for 6 h. During this labeling period, BrdU is incorporated into the DNA of cycling cells. After removing the labeling medium, the cells are fixed and the DNA is denatured in one step by adding FixDenat. After removing FixDenat, anti-BrdU-POD antibody was added to visualize BrdU that had been incorporated into newly synthesized cellular DNA. The reaction product was quantified by measuring absorbance at 370 nm in a microplate reader. For the MTT assay, MTT reagent (5 mg/ml; Invitrogen) was added directly to each well and then incubated for 4 h at 37 °C. The MTT reaction was terminated by the addition of DMSO and cell viability was assessed by a microplate reader at 490 nm.

Results

Beneficial effects of C21 was observed on human pancreatic progenitor cells (PPCs), which served as a stem cell model to study the property of cell differentiation and

regeneration. The inventors demonstrated that AT ₂ R activation induced protection of human PPCs from streptozotocin (STZ) via increased cell proliferation and reduction of oxidative stress.

STZ evoked a dose-dependent reduction in the viability of PCCs, as shown by MTT assay data, compared to Control cells (Fig. 2A), whereas C21 pre-conditioning alone did not affect cell viability (Fig. 2B). However, 1 μΜ and 10 μΜ C21 pre-conditioning resulted in improved cell viability (by 20-30%) in STZ-exposed cells, versus STZ only cells. This protective effect of C21 was abolished by AT ₂ R antagonism with 10 μΜ PD123319 (Fig. 2C).

Oxidative stress, as reflected by DHE intensity, in STZ-treated cells was markedly reduced in the cells pre-conditioned with C21 before STZ exposure (Fig. 2D), and the C21 effect was again abolished by 10 μΜ PD123319 (Fig. 2D and 2E). Similarly, SOD1 mRNA levels were significantly down-regulated in STZ-treated cells, and this down-regulation was reversed by pre-conditioning with C21 (Fig. 2F).

The DHE staining results and the MTT assay results were consistent with the possibility that the protective mechanism of C21 's effects may involve proliferative and anti-oxidative pathways.