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Title:
DERIVATIVES FOR INDUCING OSTEOGENIC DIFFERENTIATION
Document Type and Number:
WIPO Patent Application WO/2012/115575
Kind Code:
A1
Abstract:
The present invention relates to a compound for use as a medicament, especially for treating bone, tooth and cartilage related diseases and damages. The compound has shown to be inducing and enhancing osteogenic differentiation. The present invention further relates to implants comprising said compound.

Inventors:
GRANELI CECILIA (SE)
KARLSSON CAMILLA (SE)
LINDAHL ANDERS (SE)
THOMSEN PETER (SE)
Application Number:
PCT/SE2012/050180
Publication Date:
August 30, 2012
Filing Date:
February 20, 2012
Export Citation:
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Assignee:
BIOMATCELL AB (SE)
GRANELI CECILIA (SE)
KARLSSON CAMILLA (SE)
LINDAHL ANDERS (SE)
THOMSEN PETER (SE)
International Classes:
A61K31/53; A61K31/505; A61P19/10; C07D251/70; C07D471/00; C07D487/04
Domestic Patent References:
WO2000049018A12000-08-24
WO2004035132A22004-04-29
WO1999036410A11999-07-22
Other References:
ZHENG M. ET AL.: "Synthesis and antitumor evaluation of a novel series of triaminotriazine derivatives", BIOORGANIC & MEDICINAL CHEMISTRY, vol. 4, no. 15, 2007, pages 1815.
KAPIL ARYA ET AL.: "Synthesis and cytotoxic activity of trisubstituted-1,3,5-triazines.", BIOORGANIC & MEDICINAL CHEMISTRY LETTERS, vol. 24, no. 12, 2007, pages 3298.
Attorney, Agent or Firm:
BRANN AB (S- Stockholm, SE)
Download PDF:
Claims:

wherein each R4 to Re are independently an alkyl group preferably a methyl, ethyl, propyl, butyl or pentyl group, or stereoisomers or pharmaceutically acceptable salts thereof; for use as a medicament. 2. A compound according to claim 1 selected from

wherein each R4 to Re are independently an alkyl group preferably a methyl, ethyl, propyl, butyl or pentyl group; or stereoisomers or pharmaceutically acceptable salts thereof.

3. A compound according to any one of claims 1 and 2 wherein each R4 to Re are independently methyl or ethyl groups.

4. A compound according to any of the preceding claims wherein the compound is selected from the group consisting of compounds C, D, H, I and J,

or stereoisomers or pharmaceutically acceptable salts thereof.

5. A compound according to any of the preceding claims wherein the compound is selected from the group consisting of compounds D, H and J.

6. A compound according to formula 1

wherein

X is carbon or nitrogen; each Ri, R and R3 are independently selected from the group consisting of

OCH(CF3) , -CF3, -O-CF3, or hydrogen, or when X is a carbon Ri, R2 or R3 may form a penta or hexa cyclic group with said carbon comprising at least one nitrogen atom wherein at least one nitrogen is substituted with an alkyl, phenyl and /or a benzyl group; and least one of Ri, R2 or R3 is

or

the compound is selected from

or

wherein each R4 to Re are independently an alkyl group preferably a methyl, ethyl, propyl, butyl or pentyl group, or stereoisomers or pharmaceutically acceptable salts thereof; for use in a medicament for treating bone, tooth and cartilage related diseases or damages.

7. A compound according to claim 6 wherein each Ri, R2 and R3 are independently selected from the group consisting of

, -OCH(CF3)2, -CF3, -O-CF3, or hydrogen, or when X is a carbon Ri, R2 or R3 may form a penta or hexa cyclic group with said carbon comprising at least one nitrogen atom wherein at least one nitrogen is substituted with an alkyl, phenyl and/or a benzyl group; and one of Ri, R2 or R3 is

wherein each R4 to Re are independently a methyl, ethyl, propyl, butyl or pentyl group, or stereoisomers or pharmaceutically acceptable salts thereof.

8. The compound according to claim 6 wherein the compound is selected from

wherein each R4 to Re are independently an alkyl group preferably a methyl, ethyl, propyl, butyl or pentyl group, or stereoisomers or pharmaceutically acceptable salts thereof.

9. The compound according to any one of claims 6 to 8 wherein each R4 to Rs are independently methyl or ethyl groups.

10. The compound according to any one of claims 6 to 9 wherein the compound is selected from the group consisting of compounds A, C, D, H, I and J,

or stereoisomers or pharmaceutically acceptable salts thereof.

11. A compound according to any one claims 6 to 10 wherein the compound selected from the group consisting of compounds A, D, H and J.

12. The use of a compound as defined in any one of claims 1 to 1 1 to induce or enhance osteogenic differentiation of mesenchymal stem cells.

13. The use of a compound as defined in any one of claims 1 to 11 to enhance bone growth.

14. The use of a compound as defined in any one of claims 1 to 11 to induce or enhance adipogenic differentiation. 15. The use according to claim 14 wherein the compound is compound C.

16. A drug delivery vehicle containing a compound as defined in any one of claims 1 to 11. 17. A medicament comprising a compound according to any one of claims 1 to 1 1.

18. A pharmaceutical composition comprising a compound as defined in any of claims 1- 11 , and a suitable excipient or adjuvant. 19. A pharmaceutical composition according to claim 18 wherein the compound is present in a pharmaceutically active amount.

20. A method of treating a bone, tooth or cartilage defect or damage by applying a compound as defined in any one of claims 1 to 1 1 to said defect or damage.

21. An implant comprising a compound as defined in any one of claims 1 to 1 1.

22. The implant according to claim 21 wherein the implant is coated with the compound.

23. The implant according to any one of claims 21 and 22 wherein the implant is soaked with the compound.

24. A compound as defined in any one of claims 1 to 1 1 for the treatment of mesenchymal stem cells to induce or enhance osteogenic differentiation.

25. A compound as defined in any one of claims 1 to 11 for the treatment to enhance bone growth.

26. A compound as defined in any one of claims 1 to 11 for the treatment of osteoblastic progenitor cells or osteoblasts to enhance matrix production and mineralization .

27. A compound according to any one of claims 24 to 26 wherein the compound is selected from the group consisting of A, D, H or J.

28. A compound as defined in any one of claims 1 to 1 1 for the treatment to induce or enhance adipogenic differentiation.

29. The compound according to claim 28 wherein said compound is compound C.

Description:
Derivatives for inducing osteogenic differentiation

FIELD OF TECHNOLOGY

The present invention relates to osteoinductive substances, compositions comprising the same and the use of said substances and compounds and compositions.

BACKGROUND

Mesenchymal stem cells (MSCs) have been described extensively in literature as a putative cell type for tissue engineering, cell therapy and as an in vivo target for pharmaceutical and biological modulation. Its capacity to differentiate, both in vitro and in vivo to different connective tissue lineages such as cartilage, muscle and bone is the main trait that makes all these scenarios possible. Treatment of bone defects, e.g. due to trauma, a bone related diseases such as osteoporosis and achieving enhanced osseointegration of orthopaedic and dental implants may involve strategies which target MSCs, osteoblastic progenitors and osteoblasts in vivo. There are several main pathways involved in osteogenic differentiation that could be potential targets for in vivo modulation of the differentiation process, among others, the bone morphogenic protein (BMP)-, Wnt- and hedgehog (Hh)- pathways.

BMPs are members of the transforming growth factor beta (TGF-β) superfamily and have many interaction partners in the osteogenic differentiation process. They bind to a cell surface complex consisting of bone morphogenic receptors type I and II I and II with subsequent activation of a SMAD or MAP kinase pathway resulting in upregulation of target genes such as runt-related transcription factor 2 (Runx2).

Hh signalling is essential for many processes during development in vertebrates. It has also been shown that the activation of this signalling pathway can increase osteogenic differentiation of MSCs, both in vitro and in vivo. One of three different Hh ligands, sonic Hh (SHh), indian Hh (IHh) or desert Hh (DHh) binds the Patched (Ptc) / Smoothened (Smo) cell surface complex and thereby allows for the activation of transcription factors glioma-associated oncogene homologs (Gli) 1 , 2 or 3 and subsequent regulation of target genes.

These signalling pathways are possible targets for studies of MSC differentiation. For example, clinical trials are currently ongoing where recombinant BMP-2 and -7 are used in spinal fusions as a treatment for degenerative discs and back pain. This has developed from many years of research on the effects of BMP both in vitro and in vivo. However, as a large-scale clinical solution these have their limits especially in cost and administration, and a small molecule substance could be a better alternative.

By screening a chemical library of over 50,000 substances Wu et al. (J Am Chem Soc 124(49): 14520-14521) found a 2,6,9-trisubstituted purine named

purmorphamine. This substance was described as an osteogenesis-inducing small molecule substance, on the basis of increased alkaline phosphatase (ALP) activity in C3H 10T 1 / 2 cells and increased Runx2 expression in various mouse cell lines (J Am Chem Soc, 124, (49): 14520- 14521). Purmorphamine has also been shown to induce increased ALP-activity in human MSCs (hMSCs). The mechanism by which purmorphamine induces osteogenic differentiation was revealed by micro array, where activation of the SHh signalling pathway was indicated by the up- or down-regulated expression of several of its target genes (Wu et al. Chem Biol 11 (9): 1229-38). Recently, Plaisant et al. (Stem Cells 27(3):703-713) verified that purmorphamine activates the SHh-pathway. However, their results indicate that this activation in fact leads to a decrease in osteogenic differentiation of hMSCs derived from adipose tissue.

SUMMARY OF THE INVENTION

The object of the present invention is to present one or more of a compound that enhances the osteogenic differentiation of hMSCs and the bone matrix formation of hMSCs, osteoblastic progenitors and osteoblasts.

These compounds are evaluated by a quantitative osteogenic panel determining protein expression, extra cellular matrix (ECM) mineralisation and the gene expression levels of several markers for osteogenic differentiation. One aspect of the present invention relates to a compound according to formula 1

wherein

X is carbon or nitrogen;

Ri, R2 and R3 are selected from the groups consisting of

, ,-OCH(CF 3 ) 2 , -CF 3 , -O-CF3, or hydrogen, or when X is a carbon Ri, R2 or R3 may form a penta or hexa cyclic group with said carbon comprising at least one nitrogen atom wherein at least one nitrogen is substituted with an alkyl, phenyl and/or a benzyl group; and

R 4 to Re are independently a methyl, ethyl, propyl, butyl and pentyl group, for use in a medicament.

Another aspect relates to a compound according to formula 1

R 2 1

wherein

X is carbon or nitrogen;

Ri, R and R3 are selected from the groups consisting of

, -OCH(CF 3 ) 2 , -CF 3 , -O-CF3, or hydrogen, or when X is a carbon Ri, R2 or R3 may form a penta or hexa cyclic group with said carbon comprising at least one nitrogen atom wherein at least one nitrogen is substituted with an alkyl, phenyl and/or a benzyl group; and R 4 to Re are independently a methyl, ethyl, propyl, butyl and pentyl group, for use in a medicament for treating bone, tooth and cartilage related diseases or damages.

Another aspect of the present invention relates to the use of the compound defined above to induce or enhance osteogenic differentiation of mesenchymal stem cells and to induce or enhance matrix production and mineralization of osteoblastic progenitor cells or osteoblasts.

Another aspect of the present invention relates to the use of the compound defined above to enhance bone growth.

Another aspect of the present invention relates to the use of the compound defined above to induce or enhance adipogenic differentiation.

Another aspect of the present invention relates to an implant comprising the compound defined above. Another aspect of the present invention relates to a drug delivery vehicle containing the compound defined above.

Another aspect of the present invention relates to a medicament comprising the compound.

Another aspect of the present invention relates to a method of treating a bone, tooth or cartilage defect or damage by applying the compound as defined above to said defect or damage. Yet another aspect of the present invention relates to a pharmaceutical composition comprising the compound as defined above and a suitable excipient or adjuvant.

Yet another aspect relates to a compound as defined above for the treatment of mesenchymal stem cells to induce or enhance osteogenic differentiation. Yet another aspect relates to a compound as defined above for the treatment to enhance bone growth.

Yet another aspect relates to a compound as defined above for the treatment to induce or enhance adipogenic differentiation.

Yet another aspect relates to a compound as defined above for the treatment of osteoblastic progenitor cells or osteoblasts to enhance matrix production and mineralization .

Specific embodiments of the present invention are as defined in the dependent claims which are hereby incorporated into the description.

DESCRIPTION OF THE FIGURES

Figure 1

Flow cytometry histograms and dot-plot showing the expression patterns of CD 166, CD 105, CD34 and CD45. Figure 2

Mean (A) LDH and (B) ALP activity per 10.000 cells in hMSCs cultured in osteogenic conditions for two weeks, without (ctrl) or with 1 1 different compounds . (C) Mean calcium and phosphate content of the ECM of hMSCs cultured in osteogenic conditions for five weeks, without (ctrl) or with 1 1 different compounds. Cl=2.0 μΜ and C2=5.0 μΜ. a p-value<0.05 when compared to the control.

Figure 3

(A) Mean ALP activity per cell in hMSCs cultured in osteogenic conditions for two weeks, without (ctrl) or with either purmorphamine or one of five different osteoinductive compunds. (B) Mean calcium and phosphate content of the ECM of hMSCs cultured in osteogenic conditions for five weeks, without (ctrl) or with either purmorphamine or one of five different osteoinductive compounds. Cl=0.5 μΜ, C2=l μΜ, C3=2 μΜ, C4=5 μΜ and C5=10 μΜ. p-value<0.05 when compared to the control. Figure 4

(A) Mean ALP activity per 10,000 cells in hMSCs cultured in osteogenic medium for two weeks, (B) Calcium content of the ECM of hMSCs cultured in osteogenic conditions for five weeks, without (ctrl) or with either purmorphamine or one of three different compunds. White bares - without BMP-2, Striped bares - with BMP- 2 (25ng/ml). Cl=0.5 μΜ, C2= l μΜ and C3=2 μΜ. a p-value<0.05 when compared to the control.

Figure 5

Relative gene expression quantified by qPCR for (A) Runx2, (B) OCN and (C) Glil in hMSCs cultured in osteogenic medium supplemented with BMP2 for two weeks. Osteogenic medium without (ctrl) or with either purmorphamine or one of three different compounds at a concentration of 2 μΜ.

a p-value<0.05 when compared to the control, b p-value<0.05 compared to the same substance without BMP.

Figure 6

To the right, triglyceride depositions, lipid vacuoles, in the cells treated with component C, to the left control.

DETAILED DESCRIPTION OF THE INVENTION

In the present application benzylic rings illustrated with the structure should be interpreted as having a substituent in any position on the ring.

The present invention relates to one or more compounds defined in formula 1

wherein

X is carbon or nitrogen;

-CF3, -O-CF3, or hydrogen, or when X is a carbon Ri, R2 or R3 may form a penta or a hexa cyclic group with said carbon comprising at least one nitrogen atom wherein at least one nitrogen is substituted with an alkyl, phenyl and/ or a benzyl group; and R 4 to Re are independently a methyl, ethyl, propyl, butyl and pentyl group.

It should be noted that the *-mark denotes the binding point to the aromatic ring in formula I. The present invention relates to all stereoisomers obtainable, i.e.

enantiomers, diastereomers, configurational and conformational stereoisomers.

These compounds have potential of being osteoinductive and are therefore of interest for use as a medicament, especially for the treatment of various bone, tooth and cartilage diseases and defects. Further, the compounds may also induce adipogenic differentiation.

In one embodiment of the present invention the R 4 to Re-groups are methyl, ethyl, propyl, butyl or pentyl groups but preferably they are independently methyl, ethyl or propyl groups, more preferably methyl or ethyl groups and more preferably methyl groups.

In one embodiment each Ri, R 2 and R3 are independently selected from the group consisting of -CF3, -O-CF3, or hydrogen, or when X is a carbon Ri, R2 or R3 may form a penta or a hexa cyclic group with said carbon comprising at least one nitrogen atom wherein at least one nitrogen is substituted with an alkyl, phenyl and/ or a benzyl group; and

each R 4 to Re are independently an alkyl group preferably a methyl, ethyl, propyl, butyl or pentyl group.

In one embodiment Ri, R 2 or R3 are independently selected from the substituents I, II, IV, V, VI, VII, VIII and IX defined above. When X in formula 1 is a carbon Ri, R 2 or R3 may form a penta or a hexa cyclic group with said carbon comprising at least one nitrogen atom wherein at least one nitrogen is substituted with an alkyl, phenyl and/or a benzyl group. In another embodiment at least one of Ri, R 2 and R3 is I. In one embodiment each Ri, R 2 or R3 are independently selected from I, II, V, IX, XI, XII, XIII and XIV-CF3, -O-CF3, or hydrogen, or when X is a carbon Ri, R2 or R3 may form a penta or a hexa cyclic group with said carbon comprising at least one nitrogen atom wherein at least one nitrogen is substituted with an alkyl, phenyl and/ or a benzyl group; and

each R 4 to Re are independently an alkyl group preferably a methyl, ethyl, propyl, butyl or pentyl group.

When X in formula 1 is a carbon Ri, R 2 or R3 may individually form a penta or a hexa cyclic group with said carbon comprising at least one nitrogen atom wherein at least one nitrogen is substituted with an alkyl, phenyl and/ or a benzyl group.

Preferably, only one of Ri to R3 forms said cyclic group. Further, this cyclic group is preferably a penta cyclic group. Additionally, these cyclic groups should preferably comprise two nitrogen atoms. In yet another embodiment a compound according to formula 1

1 wherein

X is carbon or nitrogen; each Ri, R 2 and R3 are independently selected from the group consisting of

OCH(CF3) , -CF3, -O-CF3, or hydrogen, or when X is a carbon Ri, R2 or R3 may form a penta or hexa cyclic group with said carbon comprising at least one nitrogen atom wherein at least one nitrogen is substituted with an alkyl, phenyl and /or a benzyl group; and least one of Ri, R 2 or R3 is

the compound is selected from

wherein each R 4 to Re are independently an alkyl group preferably a methyl, ethyl, propyl, butyl or pentyl group, or stereoisomers or pharmaceutically acceptable salts thereof; for use in a medicament for treating bone, tooth and cartilage related diseases or damages.

In one embodiment the compound is selected from

, and

wherein each R 4 to Re are independently an alkyl group preferably a methyl, ethyl, propyl, butyl or pentyl group, or stereoisomers or pharmaceutically acceptable salts thereof.

wherein each R 4 to Re are independently an alkyl group preferably a methyl, ethyl, propyl, butyl or pentyl group, or stereoisomers or pharmaceutically acceptable salts thereof; for use as a medicament.

Each R 4 to Re could be arranged in para, ortho or meta position. When there is two substituents on a ring the substituents may both be arranged in ortho or both in meta position, or one in ortho and one in meta position, or one in para and one in meta position or one in para and one in ortho position. Preferably one of the substituents should be in para position. In another embodiment the compound is selected from

wherein each R 4 to Re are independently an alkyl group preferably a methyl, ethyl, propyl, butyl or pentyl group; or stereoisomers or pharmaceutically acceptable salts thereof.

In another embodiment the compound is selected from the group consisting of compounds A, C, D, H, I and J,

A

or stereoisomers or pharmaceutically acceptable salts thereof.

In another embodiment each Ri, R 2 and R3 are independently selected from the group consisting of

, -OCH(CF 3 ) 2 , -CF 3 , -O-CF3, or hydrogen, or when X is a carbon Ri, R 2 or R3 may form a penta or hexa cyclic group with said carbon comprising at least one nitrogen atom wherein at least one nitrogen is substituted with an alkyl, phenyl and/or a benzyl group; and wherein at least one of Ri, R2 or R3 is

wherein each R 4 to Re are independently a methyl, ethyl, propyl, butyl or pentyl group, or stereoisomers or pharmaceutically acceptable salts thereof.

In one embodiment the compound is selected from the group consisting of compounds C, D, H, I and J,

D

or stereoisomers or pharmaceutically acceptable salts thereof. Specific examples of compounds of formula 1 are

Chemical formula: C16H15F6N5O3 Abbreviation: A

Chemical formula: C24H24N6O2 Abbreviation: C

Chemical formula: C22H22N6 Abbreviation: D

Chemical formula: C17H17F6N5O2 Abbreviation: H

Chemical formula: C22H24F1N7O3

Abbreviation: I

Chemical formula: C22H24F1N7O3 Abbreviation: I

Chemical formula: C20H15F3N6O2

Abbreviation: J

In one embodiment the compound is one of compounds C, D, H, I or J.

In one embodiment the compound is one of compounds A, D, H or J.

In another embodiment the compound is selected from D, H or J.

In one embodiment compound C is used to induce or enhance adipogenic differentiation.

In another embodiment compound C is used for the treatment to induce or enhance adipogenic differentiation.

In another embodiment compounds A, D, H or J are used for the treatment of mesenchymal stem cells to induce or enhance osteogenic differentiation, or for the treatment to enhance bone growth, or for the treatment of osteoblastic progenitor cells or osteoblasts to enhance matrix production and mineralization. Table 1.

The pharmaceutical compositions and medicaments according to the present invention may in a certain embodiments comprise two or more compounds, for example three or four compounds. The compound could be used as a medicament but also as a pharmaceutical composition and may then also comprise various excipients or adjuvants. In one embodiment, the compound is present in a pharmaceutical composition in a pharmaceutically active amount. The medicament could be used to treat

osteoporosis, osteogenesis imperfecta, Paget's disease of bone or to induce or enhance osteogenetic differentiation, extra cellular matrix production and or mineralization .

The pharmaceutical composition according to the present invention is for enteral, topical or parenteral administration. The composition could further comprise a suitable carrier or a capsule.

WO2004 / 0889286 describes compounds similar to the present compounds but none of the compounds described herein are explicitly mentioned and the purpose of the compounds of WO2004/ 0889286 are to treat or prevent disorders associated with abnormal or deregulated tyrosine kinase activity.

In this study 11 substances were screened for toxicity and osteoinductive capacity using an extensive quantitative osteogenic panel in hMSCs and compared to osteoinductive substance purmorphamine.

ALP activity is commonly used as a marker for osteogenic differentiation, and has previously been used in similar studies screening purmorphamine analogues in the C2C12 cell line. The present study has, in addition to ALP assays, quantitatively evaluated the substances by the more clinically relevant capacity of inducing ECM mineralization as well as by the expression of several genes connected to

osteogenesis.

The results of the present invention show that, purmorphamine and the derivatives abbreviated A, D, H and J all have osteoinductive effects on hMSCs generating ALP activity, expression of common osteogenic markers, and ultimately a more mineralised ECM than compared to the control.

Furthermore, substances A and D stand out as the compounds that result in the highest degree of calcium and/ or phosphate deposition in the ECM after five weeks when osteogenic differentiation is induced without the addition of BMP-2. The intermediate concentrations of substance D almost doubled the incorporation of calcium in the ECM compared to the control. The present results of addition of recombinant BMP-2, strongly suggest that it is only the combination of purmorphamine which provide a positive synergistic effect on the induction or enhancement of the osteogenic differentiation process. This observation is interesting also from future in vivo and clinical perspectives.

Substances A, D and J are possible substitutes to purmorphamine e.g. in vivo without the expression and secretion of BMP-2 locally. Further, in a clinical situation, if the addition of a recombinant protein would be needed, an increased treatment cost would be likely.

Without being bound by theory, the hypothesis is that the high ALP levels after two weeks of culture with purmorphamine and substance J (when no BMP-2 is added) are an indication of a more progressed i.e. faster differentiation process compared to the other substances. This in turn, makes the cells more susceptible for osteogenic stimulation by BMP-2 and the combination pushes the differentiation process further.

In this context, the method of predicting osteogenic differentiation by ALP activity is of much higher relevance when differentiation is induced and stimulated by recombinant BMP-2. This methodological finding indicate that the use of ALP activity assays as a tool for evaluating osteogenic differentiation should be used in combination with other quantitative methods of analysis.

APPLICATIONS

The present invention shows the potential of the described compositions and compounds with regard to in vitro osteogenic differentiation of hMSC. There are many possible areas of application for such compositions and compounds. Hitherto, hMSC have been cultured in the presence of purmorphamine on titanium surfaces with positive osteogenic differentiation as a result (Beloti MM et al., Biomaterials 26(20):4245-8, 2005). The observations open up the possibilities to explore the in vivo effects with purmorphamine or one of the derivatives in e.g. surface

modifications and coatings on dental or orthopaedic implants in order to enhance the process of osseointegration. Other possibilities for in vivo application are treatment of bone defects with a purmorphamine-scaffold combination or purmorphamine delivered locally with a slow release carrier. The results of the present study indicate that purmorphamine or one of the structural analogues possess potent effects on the osteogenic differentiation of hMSC. Their mode of action is exerted primarily on the SHh pathway although there are implications on involvement also of the Wnt canonical pathway but the specific targets are still unknown. In order to fully understand the mechanism behind their effect and investigate their potency in vivo, further studies are required using for example genome wide micro array analysis.

The compounds according to the invention can be used in pharmaceutical compositions or alone as a medicament. Said medicament can be used for treating various bone or tooth diseases. The medicament could be administrated in any way known to a person skilled in the art, further the compound or composition could be loaded into a drug delivery vehicle.

Further, the compound could be used to coat or soak an implant in order to enhance bone or tooth growth or to induce or enhance cell differentiation at the implant site. As an example could a ceramic or polymeric implant be coated or soaked with the compound according to the invention. When placed at the defect or damaged site the compound or composition will stimulate bone or tooth growth and thereby enhance the adherence to the surrounding tissue. This presents a strategy for treating a patient with a bone or tooth defect or damage by applying the compound or composition according to the present invention to said defect or damage. EXPERIMENTS

Purmorphamine derivative screening

A virtual screening for analogues similar to purmorphamine or the previously established osteoinductive substances #3 or #70 from Lee et al. (Mol Cells

26(4):380-6, 2008) was performed by Biognos AB. A list of candidate substances was generated, and from this list a few compounds were chosen for wet screening based on their structural and chemical relationship to purmorphamine and the two substances listed above.

Cell source and culture

Bone marrow from the iliac crest was obtained from a patient undergoing surgical spinal fusion at the Sahlgrenska University Hospital. From this biopsy, a

mononuclear cell population was isolated by density gradient centrifugation using Vacutainer CPT tubes (Becton, Dickinson and Company) prefilled with Ficoll (GE Healthcare Bio-Sciences AB) according to the manufacturer's instructions. The mononuclear cell fraction was then seeded in primaria tissue culture flasks at a density of approximately 250,000 cells/cm 2 . After 24 hours the flask was rinsed with Dulbecco's modified Eagles medium-low glucose (DMEM-LG) (Thermo Fisher Scientific) and unattached cells were discarded. The adherent cells were then expanded in DMEM-LG supplemented with 2mM L-glutamine (Invitrogen) , lx Penicillin-Streptomycin (Thermo Fisher Scientific), 10 ng/mL β-FGF (Invitrogen) and 10% fetal calf serum (FCS) (Sigma), and hMSCs were enriched through culture. During expansion cells were passaged at 80% confluency using 0.05% Trypsin with EDTA (Invitrogen) and reseeded at a density of approximately 8000 cells/cm 2 .

hMSCs from a fourteen-year-old female donor were used in this study.

Flow cytometry

The cells were characterised by flow cytometry prior to osteogenic differentiation. hMSCs were stained using PE-conjugated CD 166, APC-conjugated CD105, FITC- conjugated CD34 and PE-Cy7-conjugated CD45 or the appropriate isotype controls (Bio Legend), and thereafter analysed on the FACS ARIA III (BD Bioscience). Osteogenic differentiation

Osteogenic differentiation was induced with osteogenic medium containing dexamethasone (10 μΜ), ascorbic acid (45 mM) and -glycerophosphate (20 mM). Cells in passage five were seeded at 5000 cells/cm 2 and then kept in osteogenic conditions for up to five weeks. The medium was prepared fresh every week and changed twice a week. The osteogenic medium was supplemented with

purmorphamine or one of the compounds dissolved in DMSO (Sigma Aldrich) and frozen in suitable aliquots. The control group was treated with osteogenic medium to which only DMSO was added.

An initial in vitro screening of the osteogenic potential and toxicity of 1 1 substances was made where two different concentrations were used (2.0 and 5.0 μΜ).

Subsequently, six candidate substances with the best results in the initial in vitro screening were tested in three different concentrations (0.5, 1.0 and 2.0 μΜ) and compared to purmorphamine. Finally, the three substances displaying the most effect on the differentiation process, were chosen for investigation with a full osteogenic panel covering both gene and protein expression, ECM mineralisation as well as possible synergistic effects with human recombinant BMP-2 (R&D systems) at 25ng/ml.

ALP and LDH assays

Samples in cell culture well-plates were rinsed twice with unsupplemented culture medium (DMEM-LG) and the cells were then lysed in M-PER (Thermo Fisher Scientific). The ALP activity in the cell lysate was subsequently measured using p- nitrophenylphosphate as substrate. The quantity of p-nitrophenylphosphate, which has an absorbance maximum at 405 nm, was considered directly proportional to the ALP activity. The LDH activity in the cell culture medium, used as an indication of toxicity of the compounds on cells, was analysed using a Cytotoxicity Detection Kit where the culture medium was collected and incubated with the substrate mixture from the kit. The LDH activity was subsequently determined in a coupled enzymatic reaction, during which nicotinamide adenine dinucleotide (NAD + ) is reduced to NADH. The rate of NADH increase is proportional to the catalytic activity of LDH and can be measured photometrically at 340nm.

The analyses described above were performed at the accredited laboratory at Sahlgrenska University Hospital.

RNA isolation

RNA was isolated from the samples by RNeasy micro kit (Qiagen) and possible genomic DNA contaminations were removed by DNase treatment (Applied

biosystems) according to the manufactures' protocol. A total of 150 ng RNA was transcribed to cDNA using TaqMan Reverse Transcription reagents and random hexamer primers (Applied Biosystems).

Gene expression analysis

Real-time PCR was performed with cDNA equivalent to 2,5 ng RNA and the TaqMan Universal PCR master mixture with l x assay-on-demand mixes of primers and TaqMan MGB probes. All samples were analysed in duplicates and PCR was performed using the 7900HT real time PCR System (Applied Biosystems). The assays used in this study were: Runx2, Osteocalcin (OCN), Glil and 18s as an endogenous control. The relative quantification of the target gene expression was performed according to the standard curve protocol and calculated by the ΔΔ-Ct method.

Calcium and Phosphate assay

Samples in the cell culture well-plates were rinsed twice with unsupplemented culture medium (DMEM-LG) and fixated in Histofix for 30 minutes. After rinsing the wells with distilled ¾0 the samples were demineralised by incubation in 0.6M HCl on an orbital shaker for 24 h, followed by collection of the samples and

measurement of the calcium and phosphate concentrations.

The calcium levels were measured using the ortho-cresolphthalein complexone (OCPC) method. Under alkaline conditions, this reagent forms a complex with calcium that can be detected at 600 nm. The absorbance is directly proportional to the calcium concentration. Further, the phosphate levels were measured by colometry of phospho-vanado-molybdic acid. This reagent form, under acidic conditions, a complex that can be detected at 340 nm and the absorbance is directly proportional to the phosphate concentration. The analyses described above were performed at the accredited laboratory at Sahlgrenska University Hospital.

Statistical analysis

All statistical analysis was performed in SPSSStatisticsl7.0 and non-parametric test were used since a normal distribution of the data could not be guaranteed. All groups were first compared by a Kruskal-Wallis test and if a significant difference was found all groups were compared to the control condition using a Mann- Whitney U test. A significant difference was defined as a p-value of <0.05.

Results

Purmorphamine derivative screening

From two lists of 1018 and 51 compounds, generated by the virtual screening, 1 1 substances were chosen manually. One of the substances, purvalanol (pul), was registered as an experimental drug in Drugbank database (www.drugbank.ca), where chemical substances are registered with their potential targets. However in this case, no previous literature could be found on its function in osteogenic differentiation or bone regeneration. None of the other 10 compounds were previously described. For simplicity, each compound has been given a one-letter code that can be found in Table 1 and these will be used throughout this paper.

Flow cytometry

Before the osteogenic differentiation was induced the hMSCs were analysed by flow cytometry in order to verify the mesenchymal stem cell phenotype. Approximately 98% of the cells stained positive for CD166 and CD 105, and 99% were CD34 and CD45 negative (Figure 1).

Initial screening

LDH and ALP activity were measured after two weeks of osteogenic culture, and calcium and phosphate after five weeks with concentrations of 2.0 and 5.0 μΜ of each substance.

Only the higher concentration of substance D resulted in LDH-levels indicating toxicity, which was also seen morphologically in the cell culture wells (data not shown). The other substances were all within the normal range of LDH per cell during osteogenic differentiation, although slightly elevated compared to the control, which was only treated with the DMSO vehicle throughout this study (Figure 2A). Interestingly, substance C induced adipogenic differentiation indicated by extensive lipid vacuole formation, already after two weeks (Figure 6).

In the initial screening step substances A, H, I and J resulted in higher ALP levels per cell compared to the control (Figure 2B), and substances A, D, H and J either had higher content of calcium or phosphate, or both, in the ECM (Figure 2C). For all the substances with higher ALP or calcium / phosphate levels compared to the control the lower concentration of 2 μΜ resulted in relatively higher levels than 5 μΜ with the exception of substance J for which 5 μΜ gave slightly higher levels of calcium and phosphate.

Concentration optimization

Five substances were chosen based on the results described above. Compounds A, D, H, I were added to the cells at concentrations of 0.5, 1.0 and 2.0 μΜ and substance J at 0.5, 1.0, 2.0, 5.0 and 10.0 μΜ. In this candidate evaluation the substances were compared to purmorphamine at concentrations of 0.5, 1.0 and 2.0 μΜ.

The 10.0 μΜ concentration of compound J was too high and resulted in

significantly elevated LDH levels per cell and morphological changes of the cells indicating toxicity (data not shown). Substances I, J and purmorphamine resulted in higher ALP activity compared to both the control and the other compounds. This increase was 63 % for the intermediate concentration of I. However, ultimately only compound I had equal or lower calcium and phosphate content of the ECM than the control after five weeks (Figure 3). Treatment of hMSCs for five weeks with 1.0 μΜ of substance D increased the calcium and phosphate content of the ECM by 96 and 38% respectively whereas treatment with purmorphamine resulted in a 65 and 17% increase. Treatment with 0.5 μΜ of substance H generated a large increase in calcium deposition but since only a small increase in phosphate content was detected the effect of this compound on hMSC differentiation and ECM

mineralisation is not clear. The reverse relationship between concentration and mineralization for this substance suggests that this could be a dose related issue. In depth evaluation

Based on the high degree of mineralisation detected by the addition of substances A, D, J, these compounds were evaluated in a full osteogenic panel with and without the addition of BMP-2.

The ALP levels of compounds A, D, J and purmorphamine in combination with BMP-2 are shown in Figure 4A. Interestingly, with the addition of BMP-2 ALP levels decreased significantly for all conditions and compared to the control all substances except purmorphamine resulted in a significantly lower ALP activity. Subsequent analysis of the ECM mineralisation showed that only purmorphamine had synergistic effects in combination with BMP-2 and that substances A and D had a decreased effect on the osteogenic differentiation in combination with BMP-2 (Figure 4B). A tendency to synergistic effects between BMP-2 and substance J can also be seen in the quantification of the ECM mineralization.

In order to gain insight into the mechanism behind this effect qPCR analysis was performed (Figure 5). In contrast to the ALP and ECM mineralisation results, addition of BMP-2 resulted in slightly increased runx2 expression for all substances compared to the control, with statistical significance for substances D and J. Here, the runx2 expression increased with the addition of BMP-2 when compared to unsupplemented culture for all substances except A.

The later marker of OCN had an expression pattern more in line with the

mineralisation results. The OCN expression was significantly lowered with the addition of BMP-2 for all culture conditions. However, the expression of OCN in

BMP-2 stimulated hMSCs treated with, D, J and purmorphamine was higher than the control condition.

Purmorphamine had the highest expression of SHh marker Gli 1 compared to all other conditions. The synergistic effect of BMP-2 on Hh-activation was, however, only significant in combination with substances A, D and J.