WO1992004379A1

WO1992004379A1 - A new growth factor isolated from porcine spleen

Info

Publication number: WO1992004379A1
Application number: PCT/EP1990/001565
Authority: WO
Inventors: Walter Dittrich; Axel Willig; Peter Jaros
Original assignee: Pharma Biotechnologie Hannover Gmbh
Priority date: 1990-09-03
Filing date: 1990-09-14
Publication date: 1992-03-19
Also published as: AU6404190A

Abstract

A growth factor is described which is obtainable from spleen or lung hole organ or tissue by a process comprising treatment of the biological source with proteases, dialysis and chromatography. The factor shows a molecular weight between 5 and 15 kD and is found to be a glycoprotein. It shows synergistic effects when applied on normal cells together with hormones like insulin or endogenous factors like various growth factors such as EGF. It is useful for application in medicine. A composition comprising an effective amount of that factor is applicable for wound healing, improved healing of broken bones, removing cysts in tumors, selection of normal fibroblasts, application in connection with treatment of cachexia and the like.

Description

A NEW GROWTH FACTOR ISOLATED FROM PORCINES SPLEEN.

The invention is related to a growth factor derived from various organs such as spleen, lung, liver, kidneys or tissue thereof.

Various growth factors are known for example platelet derived growth factor (PDGF) , epidermal growth factor (EGF) , fibroblast growth factors (FGF) and nerve growth factors (NGF) . For the time being the therapeutical utilization is handicapped by the fact that they not only stimulate the growth of normal cells but also that of tumor cells. For this reason it seems to be impossible to use especially the natural occuring factors for a use in medicine for example in the application of woundhealing in particular after surgery or a treatment of patients suffering form injuries.

Thus one object of the invention is to provide a factor which stimulates specifically normal tissue but does not stimulate the growth of tumor cells. Another object of this invention is to describe a process for the production of that particular factor. Still another object of this invention is to describe a medicament which can be utilized in a method for treatment of injuries regardless their origin.

In W. Dittrich et al. Exp. Cell Res. 188, 172 - 174 (1990) there is described a method for isolation of a mixture containing a factor derived from porcine spleen. This factor shows biological activities as a growth promoting factor for 3T3 fibroblasts. The inventors showed dose responsiveness of the factor and its effect on growth promotion of 3T3 cell is distinct of that of insulin and fetal calf serum. They discussed by chemical character¬ istics of the growth factor comparing it with factors of similar biological properties. They found additionally that the porcine spleen effect was able to stimulate growth of 3T3 cells even after boiling the fraction containing this extract in a water bath for 5 min. Also proteolytic treatment with trypsin for the duration of 4 h at 37^βC or with collagenase overnight did not destroy the bioactivity. Serveral protein determination revealed depending on the method 30% - 50% protein content related to the dry weight. Chromatography on Superose TM 12 re¬ vealed a single peak with an apparent molecular weight range of 8 - 10 kDa. They also reported that a cancer cell line P388 being widely used in screening drugs for anticancer activity was not stimulated by this factor. It is also reported that a synergistic effect is monitored when isulin and the factor is given simultaneously to a cell cultur with 3T3 cells.

A spleen derived growth factor (SDGF) obtainable by a process comprising the steps

a) treating, spleen, lung, liver, kidney and/or tissues thereof in presence of proteases, dialysis against water,

b) chromatography with ion exchange fast protein liquid chromatography (FPLC) or high pressure liquid chromatography (HPLC) with ion exchange materials and/or gel permeation chromatography on dextrane derived materials

c) or alternatively to step b) running a pol acryl- amid gel electrophoresis (PAGE) in a 10% acryl- amid 3% bisacrylamid gel and eluting the proteins in the range of molecular weight 5.000 - 15.000 ^" is able the solve the problems described herein above.

In particular the factor can be detected in spleen, lung, liver, kidney and/or tissues thereof in amounts of roughly 4 - 5 : 5 - 7 : 1 : 1 - 2, respectivly. More particularly the growth factor from porcine spleen has been isolated and partially purified. It is a potent growth factor for normal fibroblasts, but not for normal or transformed cell lines of different origin. The factor acts synergistically with insulin when tested on 3T3 fibroblasts, but not on the myeloid cell line P388. Synergistic effects occur with epidermal growth factor when tested on NRK-49F cells. The monolayer growth-pro¬ moting activity of the factor SDGF on V79 cells could be demonstrated in the presence of transferrin, non- essential a ino acids, and vitamins. The growth of V79 cells in soft agar was stimulated by the spleen derived growth factor independent of the presence of EGF, but NRK-49F, a cell line strictly dependent on an suitable support, could not be stimulated to grow in soft agar. The factor is stable against heat, acid, various pro¬ teases, sugar-cleaving enzymes, and nucleases. The bio¬ logical activity could be destroyed only by N-glycosidase F, an enzyme which cleaves N-glycosidic bindings of sugars to asparagine residues. Enrichment of the growth factor activity by a factor of 10 was achieved by anion exchange and molecular sieve chromatography. The treatment with N-glycosidic F shows that the sugar side chain of the glycoprotein is related with the function of the factor of the invention. Therefore, also a factor produced by treatment of the factor of the invention with N-glycosidase F is claimed.

Fig. 1 shows the effect of growth stimulation of a spleen

₄ derived growth factor (SDGF) of V79 cells. 10 cells were seeded in 24 well plates in DMEM + 5% fbs. After 24 h the medium was replaced by DMEM + 2% fbs and fraction D was added. Cell counting after 48 h incubation with a coulter counter revealed considerable growth stimulation in a dose dependent manner. The results reprents the mean of at least two independent experiments.

Fig. 2 demonstrates the inhibition of the spleen derived growth factor (SDGF) by suramin. The experiment was done as described in Fig. 1. The presence of 30 μg/ml suramin alone reduced the cell number by 20%, but achieved the complete inhibition of the growth-promoting activity of fraction D.

Fig. 3 shows growth of V79 fibroblasts in soft agar. 10 3 cells in a total of 1 ml were diluted in 0,3% agar in

DMEM + 10% heat-inactivated fbs and seeded onto a 0,6% basis layer (1 ml) in 6 well plates. After supplementation with the growth factor, the plates were incubated for 10 days and stained with MTT-reagent for 4 h.

Fig. 4 shows the synergistic effects of the spleen derived growth factor when applied simultaneously with EGF. NRK-49F cells were incubated in DMEM + 2% fbs with 4 ng/ml EGF or with 10 g/ml of fraction D or with both factors. After 2, 3 or 4 days of incubation three wells were counted. Synergistic effects with EGF are evident.

Fig. 5 shows the speciflty of the growth factor. 10 4 cells per well were seeded in 24 well plates in DMEM + 5% fbs. After 24 h the medium was removed by DMEM + 2% fbs and 100 μg/ml of the spleen-derived growth factor was added. P388 and YAC cells were seeded directly in RPMI

1640 + 2% fbs. After 2 days cell growth were estimated with a coulter counter and the results expressed as % of untreated control (= 100%) . All "normal" cells were established cell lines from rodents. A549 is a human carcinoma line from the lung. HEp2 is a human epidermoid carcinoma, P388 and YAC cells are myeloma or lymphoma cells from the mouse.

Fig. 6 shows the result of anion exchange chromatography. Fast protein liquid chromatography of the dialyzed spleen extract on a Mono Q column (Pharmacia) , equilibrated with A: 20 mmol/1 triethanolamin, pH 7,5. 500 μl sample (10 mg/ml) was eluted by a linear gradient from 0 to 50% solvent B: 20 mmol/1 triethanolamin, pH 7,5 + 1 mol/1 NaCl. The eluate was monitored at 254 n . Fractions (1,5 ml) of 3 runs were pooled and assayed for fibroblast growth activity. The graph shows results using V79 cells. The activity of the growth promoting material was found between 0,2 and 0,4 mol/1 NaCl. These fractions were pooled and desalted by dialysis.

Fig. 7 shows chromatographic separation with a Superose TM 12 column. Pooled fractions from anion exchange chroma¬ tography (Fig. 6) were purified with a Superose TM 12 column under acidic conditions (50 mmol/1 HC1) . Flow rate: 0,2 ml/min. Detection: UV at 214 nm.Sample size: 0,1 ml, calibrated with BSA (67 kDa) , trypsin inhibitor (23 kDa), and cytochrome C (12,5 kDa) (dashed line). Fractions of 2 ml each were collected from 3 runs, neu¬ tralized with NaOH, and 10 1 of each fraction was tested on V79 cells for growth-promoting activity.

Fig. 8 shows the result of a polyacrylamid gel electro- phoresis and the bioactivity of the respective fractions. A separating gel with 10% T and 3% C was overlayered by a staing gel (4% T, 3% C) . The protein test mixture 4 and 5 from Serva contained 7 proteins in the range from 90 to 6,5 kDa. All samples were incubated in 4% SDS, 12% glycerol (w/v) , 50 mM Tris, pH 7 and 2% (v/v) 2-mercapto- ethanol at 40°C for 1 h before application onto the gel. The anode buffer contained 0,2 mol/1 Tris, pH 8,9 and the cathode buffer contained each 0,1 mol/1 Tris and Tricine, pH 8,2 and 0,1% SDS. Electrophoresis started at 30 V constant for about 1 h and then raised to 150 V for 4 h. One part of the gel was fixed and stained with Coomassie R 250 and the other part of the gel was cut into pieces which were dialyzed against water. Gel rests were removed by centrifugation and the supernatant was tested on V79 cells.

The growth-promoting activities of the factor of the invention are reversible and restricted to normal fibro¬ blasts. But it should be noted that all cell lines so far tested have been established for many years and that the term "normal" indicates only the origin of the cells from normal tissue.

One of the main differences between normal and tumor cells is the higher dividing rate of the latter. A further acceleration of the dividing rate is probably more difficult to measure in fast growing tumor cells than in normal cells.

The growth of normal cells in soft agar has been used to define transforming growth factors. For bioassay of TGF-β, NR cells are usually used, because the growth of this cell line in soft agar is strictly dependent on this factor. NRK cells could not be stimulated to grow in soft agar in the presence of SDGF. The factor of the invention presence of SDGF is not identical to TGF-β. Furthermore, the synergistic effects of fraction D with EGF when tested with NRK-49F cells supports this because TGF-β is antago¬ nistic to EGF in monolayer growth of NRK cells (Roberts et al. 1985) .

From its biochemical and chromatographic behaviour of the factor of the invention it can be assumed that its active components are a heterogenous group of glycopeptides and that N-glycosidic bindings between asparagine residues and carbohydrates are important for the biological activity. The microheterogenity becomes visible form iso- electric focussing (IEF) and may be responsible for the rather broad peaks in the ion exchange chromatography as well as the fixation problems in SDS-PAGE. 0,1% SDS and 65 mmol/1 dithiotreitol could not destroy the biological activity.

The determination of the molecular weight revealed a single peak between 8 and 10 kDa on a Superose TM 12 molecular sieve column, when eluted with 50 mmol/1 ammonium acetate buffer at pH 7,2. After enrichment of the factor SDGF with anion exchange and subsequent elution under acidic conditions, peaks of an apparent M -range of approximately >10 , 30, 15 kDa and two smaller peaks appeared. In SDS-PAGE as used bands are visible in a molecular weight range between 90 and 7 kDa and the bio¬ logical activity is restricted to an area between 5 - 15 kD particularly below 12 kD.

The factor SDGF of the invention acts on normal fibro¬ blasts, but not on other cell types. In addition the factor is extremely stable against enzymatic digestion and acidic, basic or reducing conditions. An isolation procedure and further biological properties are demonstrated below.

In addition to the known isolation procedure described by the inventors by using anion exchange chromatography using fast protein liquid chromatography and monitoring UV at 254 or 214 nm a Mono Q column (Phar acia-LKB) and sub¬ sequently a HPLC system was applied using a weak basic DEAE-SilOO column (Serva, Heidelberg) . A molecular sieve chromatography (gel permeation chromatography) was done with a Superose TM 12 column. A chromatograph in fig. 7 together with the biological activity (insert) of the respective fractions.

The cell lines were cultured in either DMEM or RPMI 1640 with 5% fetal bovine serum (fbs) and penicillin/strepto¬ mycin (all from Seromed, Heidelberg) at 37°C in a humidi¬ fied atmoshere and 5% CO_. For experiments all cells that needed a suitable support for growth were trypsinized with 0,125% trypsin in phosphate-buffered salt solution (PBS) without calcium and magnesium, counted with a

₄ Coulter Counter (Coulter Electronics) and seeded at 10 cells per well in 24-well plates (Falcon) . 24 h later the medium was replaced by medium containing 2% fbs or 1% fbs and supplemented. Yac and P388 cells were collected by centrifugation, counted, seeded in RPMI 1640 medium with

2% fbs in the same cell density, and were supplemented as stated in the figures. Non essential amino acids (NEAA) , transferrin and vitamins were obtained from Gibco.

Soft agar assay: A 0,6% agar basis layer (1 ml) was over- laid with 10 3 cells in 0,3% agar + 4ng/ml EGF (1 ml) m

6-well plates (Falcon) and supplemented. Colonies were counted visually after 10 days incubation and subsequent staining with MTT reagent (Serva) for the last 4 h.

Electrophoresis: SDS-PAGE was performed as described by Schagger and von Jagow (1987) with 10% T and 3% C as a separating gel overlayered by a 4% T, 3% C stacking gel. As a standard the protein test mixtures 4 and 5 from Serva (Heidelberg) , containing 7 proteins in the range from 80 kDa to 6,5 kDa were used. The gels were fixed and stained with 0,04% Coomassie R 250 in 40% Methanol, 10% acid. Isoelectric focussing (IEF) was done with Servalyte Precotes 3 - 10 according to the method described by the manufacturer with the protein test mixture 9 from Serva. EGF and fibroblast growth factor (FGF) were obtained from Serva, Suramin was a kind gift from Boehringer, Mannheim, and PDGF was obtained from Paesel & Lorei, Frankfurt. Trypsin, Pepsin, Pronase E, Proteinase K, Collagenase, β-Glucuronidase, N-Glycosidase F, Lipase, Amyloglucosidase, Neura inidase, Ribonuclease A and Benzon-Nuclease were obtained from Serva, Boehringer or Merck, and incubated with the growth factor from the spleen under conditions recommended by the manufacturer for at least 4 h. The enzymatic activity was destroyed by boiling for 5 min and the remaining biological activity of the growth factor was assayed with V79 cells under standardized conditions and compared with growth factor activity after the same handling without enzymes. Iron content was measured with a commercial test kit (Merck, Darmstadt) .

A standard protocol using V79 cells as an indicator for the growth-promoting activity was developed. This cell line was preferred to 3T3 cells because of its higher sensitivity to the spleen-derived growth factor for fibro- blasts (Fig. 1) . Under standard conditions the cell numbers were determined 2 days after reduction of the serum content and supplementation with growth factor. The growth-stimulating effect was reversible; after depletion of the growth factor, the cells again showed normal growth rates when compared with untreated controls. The incubation of fraction D in the presence of NEAA, trans- ferrin, or vitamins resulted in all cases in an additional growth-promoting effect; thus, trivial effects of this kind can be excluded. The activity could not be neutralized by incubation with 65 mmol/1 dithiothreitol, 0,1% SDS or 0,1 mol/1 NaOH.

V79 cells displayed no interactions with PDGF (10 - 30 ng/ml), EGF (2 - 4 ng/ml), FGF (0,2 - 4 ng/ml) and insulin (1 - 5μg/ml) in this assay. No substances acted synergistically or antagonistically with fraction D but 30 μg/ml suramin, a substance, which is able to prevent the binding of EGF and PDGF to its receptor (Betsholtz,

C, Johnsson, A., Heldin, C. H. and estermark, B. (1986) Proc. Natl. Acad. Sci. USA 83, 6440), achieved a complete inhibition of the growth-promoting activity of fraction

D, as shown in Fig. 2.

V79 cells were able to grow in soft agar in the presence of fraction D, independent of the presence of EGF. The results of a typical experiment are outlined in Fig. 3. A maximal stimulation was achieved with 10 μg/ml of fraction D.

The growth-promoting activities of fraction D alone on monolayer growth of NRK-49F-cells were essentially the same as in the case of V79 cells, but in contrast to V79 cells this cell line showed synergistic effects with EGF (Fig. 4) , resembling the synergistic effects with insulin which we reported earlier. But soft agar growth of NRK cells could not be stimulated in the presence of 4 ng/ml EGF.

Only the normal fibroblast cell lines 3T3, V79, L929 and NRK (all from rodents) could be stimulated. The human lung carcinoma cell line A549, the epidermoid HEp2 car¬ cinoma and P388 myeloma and YAC lymphoma from mouse could not be stimulated, as shown in Fig. 5.

The spleen derived growth factor (SDGF) activity is not species-specific and not restricted to the spleen. Thus, it can be detected in mouse, rat and porcine spleens and in higher amounts also in mouse lungs.

Some aspects of the biochemical nature of the factor of the invention were studied by incubation with the enzymes listed above. None of these enzymes could destroy the growth-promoting activity, but first experiments with N-glycosidase F, which cleaves N-glycosidic bindings of carbohydrates with asparagine residues, resulted in a nearly complete destruction of the biological activity of fraction D. The hydrolysis of O-glycosidic bindings by 48 h incubation in 0,1 mol/1 NaOH + 1 mol/1 NaBH4 (Montreuil, J., Bouquelet, S., Debray, H. , Fornet, B. , Spik, G. and Strecker, G. in: Chaplin, M.F. and Kennedy, J.F.: Car¬ bohydrate Analysis a practical Approach. IRC Press Oxford (1986) 143) and subsequent dialysis again revealed a bio¬ logical active substance. The total sugar content of the factor was only around 5% when determined with the phenol/ sulphuric acid method (Chaplin, M.F. in: Chaplin, M.F. and Kennedy, J.F.: Carbohydrate Analysis a practical Approach. IRC Press Oxford (1986) 143).

A relatively high iron content of 0,6% of the dry weight was found but the iron was not responsible for the bio¬ logical activity. It could be depleted by dialysis against 0,1 mol/1 thioglycolic acid and subsequently against water, using the method described for the re¬ ductive mobilization of ferritin iron (Funk, F. , Lenders, J. P., Chrichton, R. R. and Schneider, . (1985) Eur. J. Biochem. 152, 167). This procedure achieved reduction and complete chelating of the iron without loss of the bio¬ logical activity. The same results were obtained by dialysis of fraction D against 0,1 mol/1 citric acid and subsequently against water.

The biological activity could not be precipitated or destroyed by treatment with 5% trichloracetic acid or 5% perchloric acid. Gradual ammonium sulfate precipitation was also found to be unsuitable for further purification of the factor of the invention. Enrichment of the biologi¬ cal activity per mg protein by a factor nearly tenfold by the use of anion exchange chromatography with a strong basic Mono Q column and collecting the fractions obtained between 0,2 and 0,4 mol/1 NaCl is outlined in Fig. 6. Essentially the same results were obtained, when a 20 mmol/1 triethanolamine buffer (pH 7,5) a 20 mmol/1 histidine buffer (pH 6) was used. Subsequent molecular sieve chromatography with a Superose TM 12 column under acidic conditions (50 mmol/1 HC1) separated two peaks at around 30 kDa and 15 kDa. The incomplete separation prevents the attribution of the biological activity to one of the peaks, but a tenfold increase of the activity compared to the crude extract was found in fraction 8, whereas the peak of higher MW and the smaller substances were not active (Fig. 7) . Succesive HPLC of fraction 8 using a DEAE SilOO column showed the presence of conta- minating material, but elimnated the biological activity. A separation with a Mono Q column and followed by DEAE- SilOO gave a rather broad peak despite the use of a steep gradient. The peak appeared after 0,2 mol/1 NaCl and showed biological activity, but no further enrichment. SDS-PAGE of this peak demonstrated impurities of the material.

The detection of the factor of the invention after SDS- PAGE was difficult, probably because of the fixation of the material. With fixation in 20% TCA and subsequent silver staining of the gels no bands could be detected. Fixation and staining in 40% methanol, 10% acetic acid and 0,025% Coomassie R 250 were reasonable results, and bands in the range of 50, 39, 30, 22, 15 and 7 kDa could be detected (Fig. 8) . The biological activity was eluted from the gel by dialysis and was shown to be restricted to an area below between 5 - 15 kD more particularly 12 kD. This step gave an enrichment of the biological activity by a factor of around 12, but it was shown by reversed phase HPLC that this fraction was inhomogenous. The detection problems mentioned for PAGE were also present with IEF (iso electic focussing ) , but when the lanes where loaded with up to 0,5 mg of the factor of the invention, many small bands in the range between pi 5 and pi 6 could be detected.

A pharmaceutical composition containing essentially an effective amount of the factor of the invention is useful in the treatment of woundhealing, improved healing of broken bones, encapsulation and/or encystation of tumors, selection of normal fibroblasts, application in connection with treatment of cachexia and the like. Oral administration of this glycoprotein is possible because it is resistent to proteolysis in the intestinal tract.

Additionally the factor of the invention can be used in vitro. For example in laboratory and research use such as cell cultures and the like.

Claims

What is claimed is:

1. A spleen derived growth factor (SDGF) or derivatives thereof obtainable by a process comprising the steps

a) treating minced spleen, lung, liver, kidney and/or tissues thereof in presence of proteases, dialysis against water,

c) or alternatively to step b) running a polyacryl- amid gel electrophoresis (PAGE) in a 10% acryl- amid 3% disacrylamid gel and eluting the proteins in the range of molecular weight 5.000 - 15.000.

2. Spleen derived growth factor (SDGF) according to claim 1, which is a glycoprotein.

3. Spleen derived growth factor (SDGF) according to claim 1 or 2 being resistent to proteases, such as trypsin, collagenase, proteinase K, proteinase E as well as nucleases, sugar cleaving enzymes.

4. Spleen derived growth factor (SDGF) according to anyone of claims 1 - 3 which is stable after treat¬ ment with heat or acids.

5. Spleen derived growth factor (SDGF) according to anyone of claims 1 - 4 showing a stimulation of the growth of normal cells but not a stimulation of growth of tumor cells.

6. Spleen derived growth factor (SDGF) according to anyone of claims 1 - 5 exhibiting a synergistic effect on cell growth with hormones and/or endogenous factors.

7. Spleen derived growth factor (SDGF) according to claim 6 said hormone being insulin.

8. Spleen derived growth factor (SDGF) according to claim 6 said endogenous factors being growth factors such as insulin like growth factors, transforming growth factors (TGF) , platelet derived growth factors (PDGF) , epidermal growth factors (EGF) , fibroblast growth factors (FGF) , nerve growth factor (NGF) .

9. Spleen derived growth factor (SDGF) according to anyone of claims 1 - 8 which activity on cell growth is destroyed by treatment with N-glycosidase F.

10. Process for preparing a spleen derived factor (SDGF) by treating minced spleen, spleen tissue or lung tissue with proteases and dialysis of the tissue against water, chromatography on anion exchange materials or alternatively by polyacrylamid gel electrophoresis under reducing conditions.

11. Process for preparing a spleen derived factor (SDGF) according to claim 10 wherein the polyacrylamid gel comprises a 10% acryla id and 3% bisacrylamid gel and eluting proteins in the range of molecular weight 5.000 - 15.000.

12. Process for preparing a spleen derived factor (SDGF) according to claim 10 wherein the anion exchange chromatography is followed up by a gel permeation chromatography on a dextrane type material.

13. Pharmaceutical composition comprising an effective amount of a factor according to anyone of claims 1 - 9.

14. Method of treatment of woundhealing, improved healing of broken bones, encapsulation and/or encystation of tumors, selection of normal fibroblasts, application in connection with treatment of cachexia and the like by administering and pharmaceutical composition according to claim 13.

15. Method of treatment of woundhealing, improved healing of broken bones, encapsulation and/or encystation of tumors, selection of normal fibroblasts, application in connection with treatment of cachexia and the like by oral administration of a pharmaceutical composition according to claim 13.

16. Method of treatment of woundhealing, improved healing of broken bones, encapsulation and/or encystation of tumors, selection of normal fibroblasts, application in connection with treatment of cachexia and the like by topcial application of a pharmaceutical composition according to claim 13.

17. Method of treatment of woundhealing, improved healing of broken bones, encapsulation and/or encystation of tumors, selection of normal fibroblasts, application in connection with treatment of cachexia and the like by injection of an effective amount of a pharmaceutical composition according to claim 13.

18. Use of a factor according to anyone of claims 1 - 9 for the preparation of a medicament for the treatment of woundhealing, improved healing of broken bones, encapsulation and/or encystation of tumors, selection of normal fibroblasts, application in connection with treatment of cachexia and the like.

19. Use a factor according to anyone of claims 1 - 9 for in vitro use, laboratory use such as cell culture and the like.

20. Pharmaceutical composition comprising besides an effective amount of a factor according to anyone of claims 1 - 9 additionally an amount of hormones and/ or endogenous factors or a combination thereof.

21. Pharmaceutical composition according to claim 20 said hormone being insulin.

22. Pharmaceutical composition according to claim 20 said endogenous factors being growth factors such us insulin like growth factors, transforming growth factors (TGF) , platelet derived growth factors (PDGF) , epidermal growth factors (EGF) , fibroblast growth factors (FGF) , nerve growth Factor (NGF) .

23. Composition comprising the factor of anyone of claims 1 - 9 after treatment with a sugar-cleaving enzyme such as N-glycosidase F and the sugar which was cleaved from the protein.

24. Sugar component of the factor according to anyone of claims 1 - 9.