WO2017017186A1

WO2017017186A1 - Small molecules for enhancing neuronal function

Info

Publication number: WO2017017186A1
Application number: PCT/EP2016/068004
Authority: WO
Inventors: Alexey Terskikh; Adrian Heini; Ernst Theodor Rietschel; Christian Alexander; Artur J. Ulmer; Jean-Pierre Mach; Reginald M. Gorczynski
Original assignee: Clinique La Prairie
Priority date: 2015-07-29
Filing date: 2016-07-28
Publication date: 2017-02-02

Abstract

The present invention relates to a compound selected from 5-hydroxymethyl-2-furfural (5HMF), a prodrug thereof, a derivative thereof, and a metabolite thereof, said derivative being capable of enhancing the affinity of hemoglobin to oxygen and/or being capable of increasing oxygen delivery to microcapillaries and/or sinusoids said metabolite preferably being selected from 5-hydroxymethylfuroic acid, 2,5-furandicarboxylic acid and N-(hydroxymethyl)furoylglycine, for use in a method of treating or preventing a condition characterized by a need for neurogenesis; neovascularization of the central nervous system; and/or enhanced neuronal function.

Description

Small molecules for enhancing neuronal function

The present invention inter alia provides a small moiecule based approach to increase adult hippocampal neurogenesis.

In particular, it relates to a compound selected from 5-hydroxymethyl-2-furfural (5HMF), a prodrug thereof, a derivative thereof, and a metabolite thereof, said derivative being capable of enhancing the affinity of hemoglobin to oxygen and/or being capable of increasing oxygen delivery to microcapillaries and/or sinusoids, said metabolite preferably being selected from 5- hydroxymethylfuroic acid, 2,5-furandicarboxylic acid and N-(hydroxymethyl)furoylglycine, for use in a method of treating or preventing a condition characterized by a need for neurogenesis; neovascularization of the central nervous system; and/or enhanced neuronal function.

In this specification, a number of documents including patent applications and manufacturer's manuals is cited. The disclosure of these documents, while not considered relevant for the patentability of this invention, is herewith incorporated by reference in its entirety. More specifically, all referenced documents are incorporated by reference to the same extent as if each individual document was specifically and individually indicated to be incorporated by reference.

The importance of adult hippocampal neurogenesis for cognitive function, stress resistance, and depression is well documented in rodents (Ming and Song, Neuron. 201 1 ; 70(4): 687- 702.; Snyder et al., Nature. 2011 ; 476(7361 ):458-61 ), and several lines of evidence suggest that it could play equally important roles in humans (Boldrini et al., Neuropsychopharmacology. 2009; 34(11 ):2376-89; Boldrini et al., Biol. Psychiatry. 2012; 72(7):562-71 ; Boldrini et al., Neuropsychopharmacology. 2013;38(6): 1068-77). Voluntary running and enriched environments have been shown to increase hippocampal neurogenesis in inbred mouse strains (van Praag et al., Nat Neurosci. 1999;2(3):266-70; Kempermann et al., Nature. 1997;386(6624):493-5), but curiously, this phenomenon has not been reproduced in wild-caught mice (Hauser et al., BMC Neurosci. 2009; 10:43; Klaus et al., Behav. Brain Res. 2012;227(2):340-7), reinforcing the interest in alternative approaches to promote neurogenesis. Because only a small fraction of amplifying neural precursors (ANPs) give rise to neuroblasts and mature neurons (Encinas et al., Cell stem cell. 2011 ;8(5):566-79), strategies tested to date have focused on enhancing neuronal survival (Pieper et al., Cell. 2010;142(1 ):39-51 ) or promoting neuronal differentiation (Wang et al., Cell stem cell. 2012;11 (1 ):23-35). However, the long-term effects of these approaches and the persistence of the increased neurogenesis remain unclear. Thus, although induced increases in symmetric or asymmetric divisions of stem cells will not accelerate their loss, recruitment of extra stem cells in the process of division may prematurely deplete the stem cell pool, with a correspondingly premature reduction in neurogenesis over the long term. Therefore, to robustly augment neurogenesis while avoiding exhaustion of the stem cell pool, it is desirable to augment self-renewal of radial glial stem cells.

The vasculature is an intrinsic component of the stem cell niche in the subgranular zone (SGZ) of the dentate gyrus (DG) (Quaegebeur et al., Neuron. 2011 ;71(3):406-24). Since the number of blood vessels in the brain declines with age (Farkas and Luiten, Prog Neurobiol. 2001 ;64(6):575-611 ), promoting vascularization of the neurogenic niche may provide a viable strategy for increasing neurogenesis (Katsimpardi et al., Science. 2014;344(6184):630-4; Licht et al., Proc Natl Acad Sci U S A, 201 1 ;108(12):5081-6).

Kato et al. (Blood. 2013;122(21 )) describe 5-hydroxymethyl-2-furfural for treatment of sickle cell anaemia.

An objective underlying the present invention is to provide novel means and methods for enhancing neuronal function as well as for treating or preventing disorders where such enhancement is desired.

Accordingly, in a first aspect, a compound selected from 5-hydroxymethyl-2-furfural (5HMF), a prodrug thereof, a derivative thereof, and a metabolite thereof, said derivative being capable of enhancing the affinity of hemoglobin to oxygen and/or being capable of increasing oxygen delivery to microcapillaries and/or sinusoids, said metabolite preferably being selected from 5- hydroxymethylfuroic acid, 2,5-furandicarboxylic acid and N-(hydroxymethyl)furoylglycine, for use in a method of treating or preventing a condition characterized by a need for neurogenesis; neovascularization of the central nervous system; and/or enhanced neuronal function.

The below formulae (1 ) to (4) represent 5HMF as well as the three recited preferred metabolites in the recited order.

The term "prodrug" is known in the art as are derivatization options for converting a compound into a prodrug. The term "prodrug" also extends to prodrug forms of said metabolites. An example is the esterification of the hydroxy group of 5HMF with C1 to C4 alkanoic acids, preferably acetic acid. The aldehyde group in compound (1 ) may be protected, as known in the art, by reacting it with 1 ,2-alkanediols or 1 ,3-alkanediols such as 1 ,2- ethanediol or 1 ,3-propanediol. C1 to C4 alkanols may be used to esterify the carboxylates in compounds (2) to (4) above. Amino group containing moieties may be used instead of alkanols. The carboxylates as present in the preferred metabolites may be esterified, preferably with C1 to C4 alkanols, such as ethanol.

Even though the term "prodrug" is used herein, this does not imply that 5HMF can only be used for therapeutic or preventive purposes. To the contrary, and this will become apparent further below, non-therapeutic uses are also envisaged. Accordingly, instead of the term "prodrug", such compounds may be referred to as inactive or less active precursors. Upon delivery, prodrugs/precursors are processed to yield the active form. In case of prodrugs/precursors being esterified versions of the active form, such processing comprises hydrolysis of the ester. The term "derivative" defines a compound which shares a structural element with 5HMF, e.g. the furan ring. A derivative of 5HMF in accordance with the present invention is further characterized in that it is capable of enhancing the affinity of hemoglobin to oxygen and/or capable of increasing oxygen delivery to microcapillaries and/or sinusoids.

The term "metabolite" has its art-established meaning. It relates to compounds which are formed in the organism upon administration of the parent compound, in the present case 5HMF. Also, the term embraces compounds which, when present in the organism, may give rise to 5HMF.

The term "preventing" has its art-established meaning. Preferably, preventing in accordance with the present invention is effected in patients which have a predisposition for any of the conditions recited herein. In that sense, any administration of 5HMF, a prodrug or metabolite thereof to a healthy person who has no predisposition for any of the conditions defined herein does not amount to a preventive, let alone a therapeutic treatment.

The term "neuronal function" is not particularly limited. Enhanced neuronal function in accordance with the present invention is preferably achieved by enhanced neurogenesis. Also preferred is that said enhanced neuronal function involves elevated levels of pCREB and/or Egr1. Related thereto, enhanced neuronal function may be achieved by enhanced proliferation of neural precursors and/or enhanced neuronal differentiation of neural precursors. Preferred neural precursors are hippocampal neural precursors. Further mechanisms of enhancing in accordance with the present invention are described in more detail below.

The term "neurogenesis" has its art-established meaning and relates to the generation of neurons, preferably such that the total number of neurons, preferably in the hippocampus, increases.

The term "neovascularization" also has its art-established meaning. It refers to the new formation of blood vessels. Preferred is the new formation of blood vessels in the neurosystem, particularly preferred in the central nervous system.

The term "elevated levels" relates to mRNA levels and/or protein levels. For either type of level art-established methods of determining are at the skilled person's disposal, including, for example, quantitative PCR (qPCR) and Northern blot analysis as used in the Examples. The term "elevated" preferably refers to statistically significant elevated levels. The p-value as compared to the negative control is preferably below 0.01 , below 10^"3, 10^'4, 10^"5, 10^"6, 10^"7, 10^" ⁸, 10^~9 or 10^"10. Expressed in percentage, it is preferred that "elevated" refers to an increase of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100%, or even larger increases such as 2-fold, 5-fold, 10-fold, 100-fold, 1 ,000-fold or higher. pCREB is the phosphorylated form of CREB, a neuronal transcription factor that plays an important role in long-term memory and synaptic plasticity (Alberini, CM. Transcription factors in long-term memory and synaptic plasticity. Physiol. Rev. 89, 121-145 (2009)). pCREB is, as known in the art, a marker of neuronal signalling activity.

Egr1 is known in the art as an immediate early gene important in learning. In terms of evidence of elevated levels of pCREB and Egr1 , reference is made to Figure 7.

The inventors' surprising findings which are further detailed in the examples enclosed herewith provide evidence of a surprising role of 5HMF in the enhancement of neuronal function. In particular, it has been found that treatment of hippocampal neural precursors with 5HMF enhances cell proliferation and neuronal differentiation. Furthermore, an enhancing effect on cell proliferation in the subgranular zone of the dentate gyrus has been observed. As shown in Figure 7, effects of 5HMF can also be seen in the CA1 and CA3 subregions (Hoge J, Kesner RP, Neurobiol Learn Mem 88:225-231 (2007); Hunsaker MR, Lee B, Kesner RP; Behav Brain Res 188:310-315 (2008)) of the hippocampus which are involved in learning and memory. Not only an effect on neural stem cells and neuronal precursor cells has been observed, but furthermore an increased formation of new blood vessels. Enhanced neurogenesis was also observed at the adult stage. Evidence for the improvement of hippocampal cognitive function is also provided.

Without wishing to be bound to a specific theory, it is considered that the underlying principle is the increase of the oxygen delivery to microcapillaries and sinusoids. Such oxygen delivery has a stimulating effect on stem cells and progenitor cells. Since such an effect is specific for the blood circulation, it goes without saying that publications describing effects of 5HMF observed in cell culture will generally have no relevance for the present invention. Means and methods for determining oxygen delivery to microcapillaries and sinusoids are art-established and described, for example, in Sakadzic S, Roussakis E, Yaseen MA, Mandeville ET, Srinivasan VJ, Arai K, Ruvinskaya S, Devor A, Lo EH, Vinogradov SA, Boas DA. Nat Methods. 2010 Sep;7(9):755-9. Also without wishing to be bound to a specific theory, it is considered that the increase of oxygen delivery to microcapillaries and sinusoids is a consequence of the compounds of the invention cross-linking hemoglobin. Cross-linked hemoglobin is a form of hemoglobin which is similar to fetal hemoglobin, especially with regard to oxygen affinity. The cross-linked hemogblobin according to the invention has increased affinity to oxygen as compared to adult hemoglobin.

Accordingly, it is understood that beneficial effects of 5HMF and its metabolites and prodrugs is not confined to the field of neurogenesis where neuronal stem cells and neuronal progenitor cells are targeted. Instead, any type of stem cell or progenitor cell may be activated and caused to divide by the administration of the mentioned compounds. Preferred non-neuronal stem cells and progenitor cells are hematopoietic, muscle, skin and intestinal stem cells and progenitor cells. A key feature of all stem cell niches is the presence of microcapillaries or sinusoids. In view of the above discussed properties of the invention (increase of the oxygen delivery to microcapillaries and sinusoids), all stem cell niches (see, e.g., Oxygen in Stem Cell Biology: A Critical Component of the Stem Cell Niche. Ahmed Mohyeldin, Tomas Garzon- Muvdi, and Alfredo Quinones-Hinojosa, Cell Stem Cell 7, August 6, 2010) benefit from the invention.

It is also apparent from the above considerations that the mentioned beneficial effects are not confined to 5HMF, its prodrugs and metabolites. Instead, any compound capable of increasing the oxygen delivery to microcapillaries and/or sinusoids is able to have a positive effect on neurogenesis, neovascularization of the central nervous system, and furthermore on proliferation of stem cells and progenitor cells in general.

In molecular terms, the mentioned increase of oxygen delivery is mediated by an enhancement of the affinity of hemoglobin to oxygen. Means and methods for determining hemoglobin affinity to oxygen are known and described, for example, in Nakagawa A, Lui FE, Wassaf D, Yefidoff-Freedman R, Casalena D, Palmer MA, Meadows J, Mozzarelli A, Ronda L, Abdulmalik O, Bloch KD, Safo MK, Zapol WM. ACS Chem Biol. 2014 Oct 17;9(10):2318-25.

Therefore, any compound which is known to increase the affinity of oxygen to hemoglobin are useful in accordance with the present invention. In fact, the present inventors surprisingly discovered that increasing the hemoglobin affinity to oxygen is a way to enhance oxygen delivery to the microcapillaries and sinusoids in order to enhance cellular functions, in particular the function of stem cell niche, including increased self-renewal of tissue stem cells; amplifying progenitors and/or differentiation and/or maturation of lineage-committed cells.

Accordingly, the present invention, in a related aspect, also provides a compound which is capable of enhancing the affinity of hemoglobin to oxygen and/or capable of increasing oxygen delivery to microcapillaries and/or sinusoids for use in a method of treating or preventing a condition characterized by a need for neurogenesis; neovascularization of the central nervous system; and/or enhanced neuronal function. Exemplary further compounds in that respect are di(5-(2,3-dihydro-1 ,4-benzodioxin-2-yl)-4H-1 ,2,4-triazol-3-yl)disulfide (TD-1), N-ethylmaleimide (NE ) and diformamidine disulfide as described in Nakagawa et al. (loc. tit).

In a further related aspect the present invention provides a compound selected from 5- hydroxymethyl-2-furfural (5HMF), a prodrug thereof, a derivative thereof, and a metabolite thereof, said derivative being capable of enhancing the affinity of hemoglobin to oxygen and/or being capable of increasing oxygen delivery to microcapillaries and/or sinusoids, said metabolite preferably being selected from 5-hydroxymethylfuroic acid, 2,5-furandicarboxylic acid and N-(hydroxymethyl)furoylglycine for use in the method of treating or preventing an age-related and/or degenerative disorder. Generally speaking, any disorder may be treated which benefits from increased self-renewal of tissue stem cells; amplifying progenitors and/or differentiation and/or maturation of lineage-committed cells.

Moreover, the present invention provides a compound which is capable of enhancing the affinity of hemoglobin to oxygen and/or capable of increasing oxygen delivery to microcapillaries and/or sinusoids for use in a method of treating or preventing an age-related or and/or degenerative disorder.

The above described surprising findings provide for several beneficial applications in the field of medicine. On the other hand, they provide for non-therapeutic and non-preventive applications in healthy individuals.

Preferred medical indications are described further below.

In a preferred embodiment of any of the aspects of the invention disclosed herein (a) said neurogenesis is hippocampal neurogenesis; (b) said neovascularization is hippocampal neovascularization; and/or (c) said enhanced neuronal function involves elevated levels of pCREB and/or Egr1.

As can be seen from the above, the hippocampus is a preferred location where the compounds of the invention effect their function.

In a second aspect, the present invention provides a compound as defined above for use in a method of treating or preventing one or more conditions selected from neurodegenerative diseases, neurovascular diseases, depression, post-traumatic stress disorder (PTSD), anxiety, anhedonia, and cognitive impairment including mild cognitive impairment (MCI), said treating or preventing neurodegenerative diseases and cognitive impairment being mediated by neurogenesis and/or neovascularisation of the central nervous system.

More generally, the present invention furthermore provides a compound which is capable of enhancing affinity of hemoglobin to oxygen and/or capable of increasing oxygen delivery to microcapillaries and/or sinusoids for use in a method of treating or preventing one or more conditions selected from neurodegenerative diseases, neurovascular diseases, depression, post-traumatic stress disorder (PTSD), anxiety, anhedonia, and cognitive impairment including subjective cognitive impairment (CDI), mild cognitive impairment (MCI), said treating or preventing neurodegenerative diseases and cognitive impairment being mediated by neurogenesis and/or neovascularisation of the central nervous system.

MCI is particularly preferred.

Given that neurogenesis and/or neovascularisation of the central nervous system is a key mechanism of the invention, it is understood that the recited diseases are especially to be targeted to the extent they involve a deficiency in neurogenesis and/or neovascularisation of the central nervous system and/or benefit from enhancing neurogenesis and/or neovascularisation of the central nervous system.

Cognitive impairment is also known as dementia. In accordance with the invention, cognitive impairment and dementia may also be treated or prevented when occurring as a symptom of a neurodegenerative disease such as Alzheimer's disease. As noted above, neurodegenerative diseases are amenable to treatment with compounds of the invention, in particular to the extent they involve a deficiency in neurogenesis and/or neovascularisation of the central nervous system and/or benefit from enhancing neurogenesis and/or neovascularisation of the central nervous system. Depression or depressive disorder is a mood disorder that causes a persistent feeling of sadness and/or loss of interest. Diagnosis of depression may be established based on the World Health Organization's International Statistical Classification of Diseases and Related Health Problems (ICD-10). In the broadest sense, the forms of depression amenable to treatment in accordance with the present invention are not particularly limited.

Post-traumatic stress disorder (PTSD) may develop after a person is exposed to one or more dramatic events.

Any of the above disorders, either inherently or preferably, is further characterized in that it is amenable to treatment by means and methods which enhance neuronal function, neurogenesis and/or neovascularization, said three mechanisms preferably being further characterized as disclosed herein.

In a preferred embodiment of both first and second aspect, 5HMF is to be administered in an amount between 1 mg and 1000 mg per day and kg bodyweight, preferably between 1 mg and 100 mg per day and kg bodyweight, and more preferably between 2 and 40 or 2 and 20 mg per day and kg bodyweight (mg/kg).

In terms of routes of administration, preference is given to the oral route. This does not mean, though, that other routes such as intravenous, subcutaneous, intrathecal or into the epidural space.

In a further preferred embodiment, the above defined compound is the only pharmaceutically active agent to be used.

In an alternative preferred embodiment, one or more further pharmaceutically active agents are to be used, said further pharmaceutically active agents preferably being selected from antidepressants, mood stabilizers and antipsychotics and monophosphoryl lipid A (MPLA).

The term "monophosphoryl lipid A" designates an art-established class of compounds. In structural terms, these compounds are derivatives of a bacterial cell wall component known as lipopolysaccharid (LPS), more specifically of a portion of said LPS which portion is known as lipid A. In functional terms, lipid A derivatives have been described as adjuvants for vaccination. An early review describing the structure of lipid A is Takada and Kotani (Clinical Reviews in Microbiology, 16, 477 (1989)). More recent lipid A mimetics which also fall under the term "monophosphoryl lipid A" in accordance with the present invention are reviewed, for example, in Persing et al. (Trends in Microbiology, 10, S32 (2002)). A preferred MPLA is the followin :

This compound is available from Avanti Polar Lipids, Inc. (Alabaster, Alabama, USA), product No. 770030.

Related to the above disclosed combination therapies, it is envisaged that the above defined compound and said one or more further pharmaceutically active agents are (a) comprised in a single formulation; or (b) formulated separately.

To the extent the pharmaceutically active agents are formulated separately, said compound and said one or more further pharmaceutically active agents are to be administered concomitantly or subsequently.

Each of the above disclosed uses inherently provides for said compound being a pharmaceutical composition or being comprised in a pharmaceutical composition.

As is common in the art, the pharmaceutical composition may further comprise pharmaceutically acceptable carriers, excipients and/or diluents. Examples of suitable pharmaceutical carriers, excipients and/or diluents are well known in the art and include phosphate buffered saline solutions, water, emulsions, such as oil/water emulsions, various types of wetting agents, sterile solutions etc. Compositions comprising such carriers can be formulated by well-known conventional methods. These pharmaceutical compositions can be administered to the subject at a suitable dose. Administration of the suitable compositions may be effected by different ways, e.g., as noted above, these options being preferred, and furthermore by intraperitoneal, intramuscular, topical, intradermal, intranasal or intrabronchial administration. Formulations for oral administration include tablets and syrups. The compositions may also be administered directly to the target site, e.g., by biolistic delivery to an external or internal target site. The dosage regimen will be determined by the attending physician and clinical factors. As is well known in the medical arts, dosages for any one patient depends upon many factors, including the patient's size, body surface area, age, the particular compound to be administered, sex, time and route of administration, general health, and other drugs being administered concurrently. Preferred doses are disclosed above.

In a third aspect, the present invention provides a food supplement or enriched food comprising or consisting of the above defined compound.

Furthermore, the present invention provides a food supplement or enriched food comprising or consisting of a compound which is capable of enhancing the affinity of hemoglobin to oxygen and/or capable of increasing oxygen delivery to microcapillaries and sinusoids.

As noted above, said compounds provide beneficial effects to a healthy person, a healthy person preferably being a person without a predisposition for any of the medical indications or disorders disclosed herein above.

The food supplement is a composition which may be added to food. Alternatively, it may be dissolved in water or a drink. In that sense, the present invention also embraces a beverage comprising the above defined compound.

An enriched food is a composition which primarily has the characteristics of food, but has been treated by adding (a) compound(s) according to the present invention.

In a preferred embodiment, said enriched food comprises (a) said compound in a concentration such that daily doses of 5HMF of 2 to 40 mg/kg bodyweight, more preferably 2 to 20 mg/kg bodyweight are provided; (b) a concentration of 0.5% w/w to 20% w/w of said compound; and/or (c) roasted coffee or dried plums. Further preferred daily doses of 5HMF include 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 25, 30, 35, and 40 mg/kg bodyweight.

The actual compound comprised in said enriched food may be any one of 5-hydroxymethyl-2- furfural (5HMF), a prodrug thereof, a derivative thereof, and a metabolite thereof, said derivative being capable of enhancing the affinity of hemoglobin to oxygen and/or being capable of increasing oxygen delivery to microcapillaries and/or sinusoids, said metabolite preferably being selected from 5-hydroxymethylfuroic acid, 2,5-furandicarboxylic acid and N- (hydroxymethyl)furoylglycine. For convenience, daily doses in terms of 5HMF are given above.

The total amount of compound in said enriched food is the sum of the natural 5HMF content of said food and the amount of added compound. Generally, but not necessarily, the major contribution to the total amount will be provided by the amount of added compound. Natural product with a comparably high content of 5HMF are roasted coffee (300 to 2900 mg/kg product) and dried plums (up to 2200 mg/kg product).

To ensure that the total dose is within the intervals specified above, said enriched food (as well as said food supplement) is preferably provided together with a description indicating the recommended amount to be taken in daily. To give an example, 5HMF may be added to roasted coffee with a natural content of 0.2% w/w such that enriched coffee is obtained which contains 10% w/w 5HMF in total. Assuming that for an espresso about 7 g coffee is used and that three espressos are consumed per day, this would provide for 2.1 g 5HMF being delivered. Assuming a body weight of 75 kg, this would be a daily dosis of 28 mg/kg bodyweight, i.e., within the above disclosed interval of 2 to 40 mg/kg bodyweight. Accordingly, the exemplary enriched food which is roasted coffee with a total 5HMF concentration of 10% w/w may be accompanied with a recommendation to consume three espressos per day. Analogous calculations and recommendations can be done and provided, respectively, by the skilled person without further ado when provided with the above information.

Further preferred total concentrations of said compound in said enriched food are 0.6, 0.7, 0.8, 0.9, 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18 or 19% w/w.

In a fourth aspect, the present invention provides the use of the above defined compound for (a) enhancing hippocampal function, in particular function of the dentate gyrus; and/or (b) enhancing cognitive ability, said cognitive ability preferably being characterized by one or more of enhanced pattern separation, enhanced associative learning and enhanced memory function; and/or enhancing resistance to stress by enhancing neurogenesis and/or neovascularisation of the central nervous system in a healthy individual.

Said healthy individual is, of course, to be understood as an individual that does not suffer from any of the diseases disclosed herein. Furthermore, it is preferred that said individuai has no predisposition for one, more or all of neurodegenerative diseases, neurovascular diseases, depression, post-traumatic stress disorder (PTSD), anxiety, anhedonia, and cognitive impairment including subjective cognitive impairment (SCI) and mild cognitive impairment (MCI).

Related thereto, the present invention provides the use of a compound capable of enhancing the affinity of hemoglobin to oxygen and/or capable of increasing oxygen delivery to microcapillaries and/or sinusoids for (a) enhancing hippocampal function, in particular function of the dentate gyrus, preferably by enhancing neurogenesis; and/or (b) enhancing cognitive ability, said cognitive ability preferably being characterized by one or more of enhanced pattern separation, enhanced associative learning and enhanced memory function; and/or enhancing resistance to stress by enhancing neurogenesis and/or neovascularisation of the central nervous system in a healthy individual.

Also related thereto, the present invention provides use of one or more compounds as defined in the first aspect of the invention for enhancing neurogenesis and/or neovascularisation of the central nervous system in a healthy individual.

These aspects relate to a non-therapeutic and non-preventive use of compounds of the invention. Further non-therapeutic effects of said compounds include an increase in overall well-being of the individual.

In a preferred embodiment of the first, second and items (a) and (b) of the fourth aspect, said treating or preventing or said enhancing, respectively, is mediated by one or more of the following: (a) neurogenesis, preferably in the dentate gyrus, preferably of (i) radial glial stem cells; (ii) amplifying neural precursors; (iii) neuroblasts; (iv) mature newborn granule neurons; (b) neovascularization, preferably in the dentate gyrus; and (c) cross-linking of hemoglobin, preferably in circulating red blood cells. Otherwise, and if not expressly indicated to the contrary, any preferred embodiment disclosed herein constitutes a preferred embodiment of any of the aspects of the present invention.

In a fifth aspect, the invention provides use of one or more compounds as defined in the first aspect for increasing oxygen delivery to microcapillaries, sinusoids and/or stem cell niches, said increasing preferably being mediated by cross-linking hemoglobin and/or enhancing the affinity of hemoglobin to oxygen, preferably in circulating red blood cells.

As noted above, it is the present inventors' surprising discovery that the compounds of the invention, preferably 5HMF, are capable of increasing oxygen delivery to microcapillaries, sinusoids and stem cell niches. Stem cell niches are typically found adjacent to said microcapillaries and sinusoids. Different from the perception prevailing in the art, namely that 5HMF acts as an antioxidant, the inventors realized that 5HMF and the above disclosed related compounds have further unprecedented effects which are not related to any antioxidant properties. Said effects include the mentioned cross-linking of hemoglobin. As such, the present inventors' discovery opens an avenue for therapeutic as well as non- therapeutic applications which are directed to activating said stem cell niches and/or triggering the proliferation of the stem cells or progenitor cells located in these regions of the human or mammalian body.

In a preferred embodiment, said use is non-therapeutic and non-preventive. In further aspects, the present invention also provides:

A compound which is capable of enhancing the affinity of hemoglobin to oxygen and/or capable of increasing oxygen delivery to microcapillaries and/or sinusoids for use in a method of treating or preventing a condition characterized by a need for neurogenesis; neovascularization of the central nervous system; and/or enhanced neuronal function.

A compound selected from 5-hydroxymethyl-2-furfural (5HMF), a prodrug thereof, a derivative thereof, and a metabolite thereof, said derivative being capable of enhancing the affinity of hemoglobin to oxygen and/or being capable of increasing oxygen delivery to microcapillaries and/or sinusoids, said metabolite preferably being selected from 5-hydroxymethylfuroic acid, 2,5-furandicarboxylic acid and N-(hydroxymethyl)furoylglycine for use in the method of treating or preventing an age-related and/or degenerative disorder. A compound which is capable of enhancing the affinity of hemoglobin to oxygen and/or capable of increasing oxygen delivery to microcapillaries and/or sinusoids for use in a method of treating or preventing an age-related or and/or degenerative disorder.

As regards the embodiments characterized in this specification, in particular in the claims, it is intended that each embodiment mentioned in a dependent claim is combined with each embodiment of each claim (independent or dependent) said dependent claim depends from. For example, in case of an independent claim 1 reciting 3 alternatives A, B and C, a dependent claim 2 reciting 3 alternatives D, E and F and a claim 3 depending from claims 1 and 2 and reciting 3 alternatives G, H and I, it is to be understood that the specification unambiguously discloses embodiments corresponding to combinations A, D, G; A, D, H; A, D, I; A, E, G; A, E, H; A, E, I; A, F, G; A, F, H; A, F, I; B, D, G; B, D, H; B, D, I; B, E, G; B, E, H; B, E, I; B, F, G; B, F, H; B, F, I; C, D, G; C, D, H; C, D, I; C, E, G; C, E, H; C, E, I; C, F, G; C, F, H; C, F, I, unless specifically mentioned otherwise.

Similarly, and also in those cases where independent and/or dependent claims do not recite alternatives, it is understood that if dependent claims refer back to a plurality of preceding claims, any combination of subject-matter covered thereby is considered to be explicitly disclosed. For example, in case of an independent claim 1 , a dependent claim 2 referring back to claim , and a dependent claim 3 referring back to both claims 2 and 1 , it follows that the combination of the subject-matter of claims 3 and 1 is clearly and unambiguously disclosed as is the combination of the subject-matter of claims 3, 2 and 1. In case a further dependent claim 4 is present which refers to any one of claims 1 to 3, it follows that the combination of the subject-matter of claims 4 and 1 , of claims 4, 2 and 1 , of claims 4, 3 and 1 , as well as of claims 4, 3, 2 and 1 is clearly and unambiguously disclosed.

The figures show:

Figure 1. Treatment with 5HMF increases proliferation and neuronal differentiation of hipNPCs. A, normalized Ki67+ cell counts under proliferation conditions (FGF+EGF). B-C, Normalized MAP2+ cell counts (B) and percentage AC3+ cells (C) under neuronal differentiation conditions (forskolin+RA). Cells were treated with 10, 100, 500, 1000 μΜ of 5HMF. *p<0.05 by one-way ANOVA. Figure 2. 5HMF treatment increases proliferation in the subgranular zone of the dentate gyrus. A-B, Oxygen tension in the blood of control mice and mice transfused with 5-HMF- treated RBCs (A) or directly injected with 5HMF (B). C-D, BrdU incorporation in mice transfused with 5-HMF-treated RBCs. E, PCNA+ cells in mice directly treated with 5HMF. ^*p<0.05, ^***p<0.005 by two-tailed t-test.

Figure 3. 5HMF treatment increases the numbers of radial glial quiescent neural precursors and amplifying neural precursors. A, Representative immunostaining (Sox2, GFAP; 30μΜ sections; flattened confocal stacks) in control and 5HMF-treated animals (5 animals per group, 0 coronal sections per animal rostra-caudal coverage of the entire dentate gyrus). B- C, quantification of QNPs (B) and ANPs (C). *p<0.05, ^**p<0.01 by two-tailed t-test. Scale bar = 50 μΜ.

Figure 4. Administration of 5HMF increases vascularization in the dentate gyrus. A, Representative immunostaining (CD31 ; 30μΜ sections; flattened confocal stacks) in control and 5HMF-treated animals (5 animals per group, 10 coronal sections per animal rostra-caudal coverage of the entire dentate gyrus). B, Quantification of (A). C, Correlation between CD31 + immunostaining and number of Sox2+ cells in the SGZ of the DG. ^*p<0.05 by two tailed t-test. Scale bar = 200 μΜ. Normalized area of DCX+ cells (D) and normalized area of CD31+ blood vessels (E) in mice treated with indicated daily doses of 5HMF. Five animals/group. ^*p<0.05, ^*p<0.01 one way ANOVA and Tukey's post hoc test.

Figure 5. Administration of 5HMF increases the numbers of neuroblasts and mature newborn neurons in the dentate gyrus. A, C, Representative immunostaining (Hoechst, DCX in A, BrdU, NeuN in B; 30μΜ sections; flattened confocal stacks) in control and 5HMF-treated animals (5 animals per group, 10 coronal sections per animal rostracaudal coverage of the entire dentate gyrus). B, D, Quantification of (A, C). ***p<0.001 by two-tailed t-test. Scale bar = 50 μΜ.

Figure 6. 5HMF treatment improves novelty/pattern recognition in long-term treated mice. A, Spatial pattern recognition test (3 x10 min. habituation sessions, then test). B, 5HMF-treated mice showed increased numbers of contacts with the object in a new location. C, Location novelty recognition test (4 x10 min. habituation sessions, then test). D, 5HMF-treated mice but not control mice recognize the 45° shift in object location. *p<0.05 by repeated-measures ANOVA with post hoc comparisons (SPSS 16.0 software). Figure 7. 5HMF increases the number of Egr1 positive neurons in various regions of the hippocampus. A Administration of 5HMF increases the numbers of Egr-1 positive neurons in the DG region of hippocampus. Long term (7 months) treatment; ^***p<0.005. B, Administration of 5HMF increases the numbers of Egr-1 positive neurons in the CA1 region of hippocampus. Long term (7 months) treatment; *p<0.05. C, Administration of 5HMF increases the numbers of Egr-1 positive neurons in the CA3 region of hippocampus. Long term (7 months) treatment; ^*p<0.05, by two tailed t-test.

Figure 8. 5HMF treatment increases the levels of pCREB in the hippocampus. A, Immunostaining for pCREB in the hippocampus of controls and 5HMF treated mice. B, Quantification pCREB intensity (normalized to DAPI area). Five animals/group. ^*p<0.05, ^**p<0.01 , two-tailed t-test.

The examples illustrate the invention. Example 1

Treatment of hipNPCs with 5HMF in vitro enhances cell proliferation and neurogenesis.

Treatment of hipNPCs with low doses of 5HMF increased their proliferation when cultured under self-renewal conditions (FGF+EFG) (Fig. 1A), and increased their neuronal differentiation when treated with forskolin and retinoic acid (Fig. 1 B). This increase could be mediated in part by the reduction in apoptosis in the differentiating cultures (Fig.l C). Intriguingly, the dose-response to 5HMF treatment was bell-shaped, with higher doses having no effect or even a reverse effect on hipNPC proliferation and neurogenesis.

Example 2

Administration of 5HMF increases cell proliferation in the SGZ of the DG.

We investigated the effect of 5HMF administration on neurogenesis in young (4-month-old) mice. Administration of 5HMF increases hemoglobin's affinity for oxygen and reduces the partial pressure of oxygen in the blood (Cabrales et a!., American journal of respiratory cell and molecular biology, 2008;38(3):354-61 ; Yalcin and Cabrales, Am J Physiol Heart Circ Physiol. 2012;303(3):H271-81 ). Two modalities have been reported for treatment of mice: transfusion of 5HMF-treated RBCs (Cabrales et al., American journal of respiratory cell and molecular biology, 2008;38(3):354-61 ) and direct administration (Yalcin and Cabrales, Am J Physiol Heart Circ Physiol. 2012;303(3):H271-81 ). We tested both techniques and observed a decrease in oxygen pressure in mice treated with 5HMF by either method (Fig. 2A, B). In addition, both methods significantly increased cell proliferation within the SGZ, as measured by BrdU incorporation and PCNA staining (Fig. 2C-E). A similarly robust increase in proliferation was seen in the SGZ of 5HMF-treated mice (data not shown). Because direct administration of 5HMF was the easier and more straightforward method, it was used in all subsequent experiments. A reduction in oxygen levels has previously been shown to increase proliferation and neurogenesis in the adult DG (Zhu et al. J Neurosci. 2010;30(38): 12653-63), providing another potential mechanism of action of 5HMF in vivo (in addition to the direct effect on neural precursors described above).

Example 3

The 5 HMF- induced increase in neural stem/precursor cells is paralleled by an increase in blood vessels.

Next, we quantified QNPs and ANPs in the SGZ following extended 5HMF treatment of young mice (i.p. injections every other day for 8 weeks, 20 mg/kg). Using confocal immunofluorescence analysis, we found that the numbers of both QNPs (GFAP+, Sox2+) and ANPs (GFAP-, Sox2+) were significantly increased after 5HMF administration (Fig. 3). These results are consistent with (although not proof of) the hypothesis that 5HMF increases self- renewal of radial glial stem cells in the SGZ of the DG.

The vasculature has been proposed to be an integral part of the neural stem cell niche (Palmer et al., J Comp Neurol. 2000;425(4):479-94; Tavazoie et al., Cell stem cell. 2008;3(3):279-88), and consistent with this, hypoxia is known to induce angiogenesis, vascular remodeling (Krock et al., Genes Cancer. 2011 ;2(12):1117-33), and hippo-campal neurogenesis (Zhu et al. J Neurosci. 2010;30(38): 12653-63). We found that 5HMF administration increased the number (but not the average diameter) of blood vessels, as detected using PECAM/CD31 staining (Fig. 4A, B). Moreover, we found that the cumulative increase in neural stem/precursor cell (Sox2+) numbers in the SGZ of the DG correlates with the increase in blood vessel numbers within the DG of individual mice (Fig. 4C). These results are consistent with an intimate connection between the neurogenic niche and vasculature (Quaegebeur et al., Neuron. 2011 ;71(3):406-24). To simplify the administration of 5HMF and reduce the invasiveness of i.p. injections, we dissolved 5HMF in the drinking water. Based on an estimated consumption of ~6 ml/day/mouse (Bachmanov et al., Behav Genet. 2002;32(6):435-43), we treated 2-month- old mice with 10, 20, or 40 mg/kg/day of 5HMF and determined the effect on neuroblasts (DCX+ cells) and blood vessels in the DG. We found that 5HMF dosedependently increased the number of DCX+ cells (Fig. 4D). Surprisingly, the lowest tested dose (10 mg/kg/day) had the biggest effect on the number of blood vessels (Fig. 4E). These data provide evidence for the independent effects of 5HMF on vascularization and neurogenesis and argue against the idea that the 5HMF induced increase in blood vessels is due to hypoxia.

Example 4

5HMF treatment increases adult hippocampal neurogenesis.

We next investigated whether treatment with 5HMF affects the numbers of neuroblasts and newborn neurons in the DG. We found that administration of 5HMF to young mice (i.p. injections every other day for 8 weeks, 20 mg/kg) robustly increased the number of DCX+ neuroblasts in the DG of treated mice (Fig. 5A). To determine the number of newborn neurons, mice were pulse-labeled with BrdU (5 days) followed by a 3-week chase, and then brain sections were double-stained with NeuN and BrdU. We found that 5HMF treatment resulted in a nearly ~2-fold increase in the number of BrdU+ NeuN+ newborn neurons in the granule layer of the DG (Fig. 5B). Intriguingly, 5HMF had a much greater effect on the numbers of DCX+ neuroblasts and, especially, mature newborn neurons (NeuN+ BrdU+) than on proliferating cells or QNPs and ANPs. Taken together, these results suggest that administration of 5HMF enhanced every stage of adult hippocampal neurogenesis. This indicates that, in addition to increasing QNP and ANP proliferation and/or survival, 5HMF might also promote neuroblast survival and neuronal survival/maturation.

In addition, we have measured the level of phosphorylated CREB (pCREB), a neuronal transcription factor that plays important role in long-term memory and synaptic plasticity (Alberini, CM. Transcription factors in long-term memory and synaptic plasticity. Physiol. Rev. 89, 121-145 (2009)) in the hippocampus of mice treated with 5-HMF. We have observed increased levels of phosphorylated CREB in the DG, CA1 , and CA3 region of the hippocampus in mice treated with 5HMF. The increase in the levels of phosphorylated CREB is reminiscent of that observed in old mice after exposure to young blood through heterochronic parabiosis (Villeda et al., Nature medicine, 20, (2014)). These data demonstrate that 5HMF treatment robustly enhances neuronal activity in several regions of hippocampus consistent with our hypothesis of brain rejuvenation following 5HMF treatment.

Example 5

Long-term administration of 5HMF improves hippocampal cognitive function.

Adult hippocampal neurogenesis is required for spatial and object recognition memory (Jessberger et al., Learn Mem. 2009; 16(2): 147-54), and increased adult neurogenesis has been shown to improve hippocampal function (Sahay et al., Nature. 2011 ;472(7344):466-70). We therefore investigated the effect of long-term treatment with 5HMF on mouse behavior. For this experiment, mice (10 controls, 15 experimental) were treated with 5HMF for 7 months (from 2 to 9 months of age, i.p. injections every other day, 20 mg/kg) and then subjected to two learning tests to assess novelty/pattern recognition, as previously described (Amador- Arjona et al., The Journal of neuroscience: the official journal of the Society for Neuroscience. 201 ;31(27):9933-44). In the spatial pattern recognition test, the location of one of three objects in the cage is altered (Fig. 6A). In the more complicated location novelty recognition test, the single object in the cage is shifted from its starting position by 34° or 45° (Fig. 6C). In both tests, the number of contacts the mouse makes with the object in the new location is scored and considered to be a measure of novelty/pattern recognition (Amador-Arjona et al., The Journal of neuroscience: the official journal of the Society for Neuroscience. 2011 ;31(27):9933-44). We found that mice treated with 5HMF performed better than untreated mice in both behavioral paradigms (Fig. 6B, D). Previous work has associated better performance in these tests with higher levels of adult hippocampal neurogenesis (Amador-Arjona et al., The Journal of neuroscience: the official journal of the Society for Neuroscience. 2011 ;31(27):9933-44; Squire et al., Nature reviews. 2007;8(11 ):872-83). Taken together, our results are consistent with 5HMF inducing an increase in the number of newborn neurons in the DG.

The immediate early gene, Egri , was shown to control the selection, maturation and functional integration of adult hippocampal newborn neurons by learning (Veyrac et al., PNAS, March 18, 2013). Therefore we examined the levels of Egri in the hippocampus of animals subjected to behavioral tests with and without 5HMF treatment. We found that animals treated with 5HMF had robust increase in Egri expression in DG, CA1 , and CA3 regions of hippocampus (Figure 7D to E). The increase in the levels of Egri is reminiscent of that observed in old mice after exposure to young blood through heterochronic parabiosis (Villeda et al., loc. cit.).

These data further demonstrate that 5HMF treatment robustly enhances neuronal activity in several regions of hippocampus in particular in middle-aged animals during enhanced cognitive activity (behavioral tests) consistent with our hypothesis of brain rejuvenation following 5HMF treatment.

Example 6

5HMF treatment activates CREB in different regions of the hippocampus.

Heterochronic parabiosis, or young plasma administration, increases levels of phosphorylated (p)CREB in the DG of aged mice (Villeda et al., Nat Med.2014;20(6):659-63). In fact, activation of CREB was sufficient to enhance cognitive function in such treated mice (Villeda et al., loc.cit.). We examined CREB activation in the hippocampus of mice treated with 5HMF using immunostaining for pCREB as in (Villeda et al., loc.cit.). We found that 5HMF administration increased pCREB levels in the DG, CA1 , and CA3 regions of the hippocampus (Fig. 8), suggesting that 5H F increases neuronal signaling globally in the hippocampus. Indeed, enhancement of neuronal activity following increased delivery of oxygen into the small blood vessels is consistent with activity-dependent oxygen consumption in neurons (Mathiesen et al., J Neurosci. 2011 ;31 (50): 18327-37; Huchzermeyer et al., J Cereb Blood Flow Metab. 2013;33(2):263-71 ).

Claims

1. A compound selected from 5-hydroxymethyl-2-furfural (5H F), a prodrug thereof, a derivative thereof, and a metabolite thereof, said derivative being capable of enhancing the affinity of hemoglobin to oxygen and/or being capable of increasing oxygen delivery to microcapillaries and/or sinusoids, said metabolite preferably being selected from 5-hydroxymethylfuroic acid, 2,5-furandicarboxylic acid and N-(hydroxymethyl)furoylglycine, for use in a method of treating or preventing a condition characterized by a need for neurogenesis; neovascularization of the central nervous system; and/or enhanced neuronal function.

2. The compound for use of claim 1 , wherein

(a) said neurogenesis is hippocampal neurogenesis;

(b) said neovascularization is hippocampal neovascularization; and/or

(c) said enhanced neuronal function involves elevated levels of pCREB and/or Egr1.

3. A compound as defined in claim 1 for use in a method of treating or preventing one or more conditions selected from neurodegenerative diseases, neurovascular diseases, depression, post-traumatic stress disorder (PTSD), anxiety, anhedonia, and cognitive impairment including subjective cognitive impairment (SCI) and mild cognitive impairment (MCI), said treating or preventing neurodegenerative diseases and cognitive impairment being mediated by neurogenesis and/or neovascularisation of the central nervous system.

4. The compound for use of any of the preceding claims, wherein said compound is to be administered in an amount between 1 mg and 1000mg per day and kg bodyweight, preferably between 1mg and 100mg per day and kg bodyweight, and more preferably between 2 and 20mg per day and kg bodyweight.

5. The compound for use of any of the preceding claims, wherein said compound is the only pharmaceutically active agent to be used.

6. The compound for use of any one of claims 1 to 4, wherein one or more further pharmaceutically active agents are to be used, said further pharmaceutically active agents preferably being selected from antidepressants, mood stabilizers and antipsychotics and monophosphoryl lipid A (MPLA).

7. The compound for use of claim 6, wherein said compound and said one or more further pharmaceutically active agents are

(a) comprised in a single formulation; or

(b) formulated separately.

8. The compound for use of claim 7(b), wherein said compound and said one or more further pharmaceutically active agents are to be administered concomitantly or subsequently.

9. A food supplement or enriched food comprising or consisting of one or more compounds as defined in claim 1.

10. The enriched food of claim 9, wherein said enriched food comprises

(a) said compound in a concentration such that daily doses of 5HMF of 2 to 40 mg/kg bodyweight, more preferably 2 to 20 mg/kg bodyweight are provided;

(b) a concentration of 0.5% w/w to 20% w/w of said compound; and/or

(c) roasted coffee or dried plums.

11. Use of one or more compounds as defined in claim 1 for enhancing neurogenesis and/or neovascularisation of the central nervous system in a healthy individual.

12. Use of one or more compounds as defined in claim 1 for

(a) enhancing hippocampal function, in particular function of the dentate gyrus; and/or

(b) enhancing cognitive ability, said cognitive ability preferably being characterized by one or more of enhanced pattern separation, enhanced associative learning and enhanced memory function; and/or enhancing resistance to stress by enhancing neurogenesis and/or neovascularisation of the central nervous system in a healthy individual.

13. The compound for use of any of claims 1 to 8 or the use of claim 12, wherein said treating or preventing or said enhancing of item (a) or (b) of claim 2, respectively, is mediated by one or more of the following: (a) neurogenesis, preferably in the dentate gyrus, preferably of

(i) radial glial stem cells;

(ii) amplifying neural precursors;

(iii) neuroblasts;

(iv) mature newborn granule neurons;

(b) neovascularization, preferably in the dentate gyrus; and

(c) cross-linking of hemoglobin, preferably in circulating red blood cells.

14. Use of one or more compounds as defined in claim 1 for increasing oxygen delivery to microcapillaries, sinusoids and/or stem cell niches, said increasing preferably being mediated by cross-linking hemoglobin and/or enhancing the affinity of hemoglobin to oxygen, preferably in circulating red blood cells.

15. The use of any one of claims 11 to 14, wherein said use is non-therapeutic and non- preventive.

16. A compound which is capable of enhancing the affinity of hemoglobin to oxygen and/or capable of increasing oxygen delivery to microcapillaries and/or sinusoids for use in a method of treating or preventing a condition characterized by a need for neurogenesis; neovascularization of the central nervous system; and/or enhanced neuronal function.

17. A compound selected from 5-hydroxymethyl-2-furfural (5HMF), a prodrug thereof, a derivative thereof, and a metabolite thereof, said derivative being capable of enhancing the affinity of hemoglobin to oxygen and/or being capable of increasing oxygen delivery to microcapillaries and/or sinusoids, said metabolite preferably being selected from 5-hydroxymethylfuroic acid, 2,5-furandicarboxylic acid and N- (hydroxymethyl)furoylglycine for use in the method of treating or preventing an age- related and/or degenerative disorder.

18. A compound which is capable of enhancing the affinity of hemoglobin to oxygen and/or capable of increasing oxygen delivery to microcapillaries and/or sinusoids for use in a method of treating or preventing an age-related or and/or degenerative disorder.