NEW MEDICINAL USE OF IGF-II

Abstract: The use of IGF-II for preparing a medicament for the regeneration of muscle tissue; and methods for medical treatment for the purpose of regenerating muscle tissue, for minimizing scar formation during healing of a wound or for preventing undesired tissue adhesion after injury, such as after surgery.

Description: NEW MEDICINAL USE OF IGF-II The present invention relates to a new medicinal use, more particularly the use of insulin-like factor II C IGF-I I) for preparing a medicament for regeneration of muscle tissue. Insulin-like growth factors, IGF-I and II, are two peptides which have been originally isolated from human se- rum. The designation IGF has been chosen due to the fact that the peptides exhibit insulin-like effects and also that their structure is homologous to that of insulin. In spite of simi¬ larities between IGF-I and IGF-II they behave physiologically quite differently. Thus, it has been found that pituitary growth hormone, GK, acts on sceletal tissues by inducing the formation of a growth factor, somatomedin, circulating in the blood and acting on the peripheral tissue. This is not the case with regard to IGF-II which is not under the same con¬ trol of GH, and the physiological role of IGF-II is still far from clear. Insulin-like growth factor II (IGF-II) is structurally significantly different from IGF-I and little is known about its therapeutic usefulness. IGF-II is a single-chain peptide containing 67 amino acid residues. For further details con- cerning IGF-II see Eur.J.Biochem. 190, 445-462 (1990), Rene E. Hu bel, Review Insulin-like Growth Factors I and II. IGF- -II and IGF-I acts via different receptors. The receptor for IGF-II is a single chain receptor with a molecular weight of 260 kD and it is identical with the mannose-6-phosphate re- ceptor (Morgan et al. Nature 329, 301-307 (1987); Kiess et al., J.Biol.Chem. 263, 9339-9344 (988). The IGF-II receptor has no homology with the IGF-I receptor which is dimeric and shows protein kinase activity. IGF-II may crossreact with the IGF-I receptor but IGF-I does not crossreact with the IGF-II receptor (Roth, R.A. Science 1988, 239, 1269-1271). In connection with extensive research in experimenta¬ tion it has now surprisingly been found that IGF-II effec¬ tively promotes regeneration of muscle tissue, whereas IGF-I does not. Thus, experiments .in. vivo on rats show that IGF-II signi icantly promotes regeneration of muscle tissue thus making the peptide useful in the treatment of muscle tissue. A similar experiment concerning wound healing in cardiac muscle unexpectedly showed increased healing and/or decreased scar formation. This is a new finding since it is an estab- lished knowledge that cardiac tissue does not regenerate. Accordingly, it is the main object of the present in¬ vention to provide new techniques for preparing medicaments effective in improving the regeneration of muscle tissue. Another object of the invention is to provide for a medicament useful in the healing of cardiac muscle tissue. A third object of the invention is to provide techni¬ ques for preparing a medicament for use in the healing of smooth muscle tissue, such as intestinal smoth muscle tissue. Still another object is to provide a method for medi- cal treatment for the purpose of regenerating muscle tissue. Accordingly, the invention resides in the use of IGF- -II for preparing a medicament for the regeneration of muscle tissue. According to a preferred aspect of the invention such use is directed to the preparation of a medicament for use in the healing of cardiac muscle tissue. An alternative aspect of the invention resides in the use of IGF-II for preparing a medicament for use in the healing of intestinal smooth muscle tissue. The invention also resides in a method for medical treatment resulting in improved regeneration of muscle tis¬ sue. Said method includes the step of administering to a pa¬ tient in need of corresponding treatment an effective amount of IGF-II. The medicinal composition used in accord with the pre- sent invention may thus in accordance with traditional phar¬ maceutical practice be formulated for use in human or veteri- nary medicine for therapeutic purposes. Such compositions may include the active ingredient IGF-II in combination with a pharmaceutically acceptable carrier, which may be solid, se- misolid or liquid. The compositions include those in a form adapted for topical application but may also be designed for administra¬ tion through other routes. Suitable forms of the composition used in applying the techniques of the present invention include tablets, capεu- les, sirups, suspensions, solutions and forms suitable for injection or infusion. The latter forms intended for injec¬ tion or infusion are preferred. Such compositions may contain conventional pharmaceutically acceptable materials, such as diluents, binders, colours, flavours, preservatives, disinte- grates and the like in accordance with conventional pharma¬ ceutical practice in a manner well understood by those skil¬ led in the art of formulating drugs. Injectable or infusable compositions of IGF-II are particularly suitable as levels of IGF-II can occur after administration by injection or infusion at the site of muscle tissue subject to regenerative healing. A particularly pre¬ ferred site for cardiac treatment is the pericardium, and the administration can take different forms, such as infusion or implantation of a slow release formulation containing IGF-II. The administered dose of the active ingredient, IGF- -II, may vary between broad limits, but a preferred range may be between about 0.1 and about 10 g per day for a week. The dose is, of course, dependent on the ' degree and type of da¬ mage, type and condition of the patient and will be determi- ned from case to case. The present invention will be further illustrated in the following by specific examples which are not to be con¬ strued as limiting the scope of the invention otherwise than the definition in the appended claims. This illustration of the invention will be made with reference to the appended drawings, wherein: Figs, la, lb and ic illustrate sections through frozen tissue samples, Fig. la being control sample, Fig. lb sample after treatment with IGF-I and Fig. Ic sample after treatment with IGF-II; Fig. 2a is an enlarged detail of the section of Fig lc; Fig. 2b is a section through a plastic embedded sample also corresponding to Fig. lc; Figs. 3a and 3b illustrate cross-sections through plastic-embedded samples, Fig. 3a illustrating the result of light microscopy and Fig. 3b showing the result of electron microscopy; and Figs. 4a and 4b illustrate corresponding sections through tissue samples treated with IGF-I. Figs. 5a-d illustrate sections through rat cardiac muscles comparing controls with IGF-II treated samples. EXAMPLE 1 Administration of IGF-I and IGF-II on sceletal muscle. In this example the animals used were male Sprague- -Dawley rats, body weight about 225 g. The animals were anae¬ sthetized and the substances tested and the vehicle for con¬ trol, 0.1% (v/v) , bovine serum albumin in physiological sali¬ ne, were infused directly into extensor digitorum longus of the rats via a thin, PE10, catheter inserted into the muscle and connected to a minipump. The total dose given was 0.2 ml administered during one week. The total dose during said week was 30 μg of IGF-I and IGF-II, respectively. After concluded administration the treated muscles were made subject to a fixing operation. Samples from the infused area were taken, on the one hand for plastic embed¬ ment for light and electron microscopy, on the other hand for sections of frozen materials for immunohistochemistry. Results Figs, la, lb and lc illustrate sections through frozen tissue, Fig. la being a control using only vehicle but no IGF, and Figs. 2b and 2c showing corresponding sections of samples after treatment with IGF-I and IGF-II, respectively. In animals infused with only vehicle "normal" granula¬ tion tissue is formed around the catheter, said tissue con¬ taining fibroblasts, blood vesβels and some inflammatory cells (Fig. la). In animals infused with IGF-I in the same vehicle approximately the same picture is found indicating no significant effect generated by IGF-I (Fig. lb). On the other hand, in animals infused with IGF-II in the same vehicle rather small elongate cells, often poly- nuclear, are found (Fig. 3b, plastic embedded section) . The corresponding cells in cryo-section were desmin-positive, i.e. muscle cells (Figs, lc and 2a). In interpreting the results it is seen that cells ori¬ ginating from IGF-II treatment are more differentiated than cells from IGF-I treatment. EXAMPLE 2 Example 1 was repeated for comparison between IGF-II and IGF-I, a total dose of both of 30 μg being administered during one week. Figs. 3a and 3b illustrate sections of plas- tic embedded tissue after treatment with IGF-II, and Figs. 4a and 4b show corresponding sections after treatment with IGF- -I . It is seen from the results obtained that IGF-II acts differently and more efficiently than does IGF-I. It is seen that IGF-II results in a relatively homogeneous population of desmin-positive cells adjacent to the catheter, and said cells have a tendency to arrange themselves in bundles rela¬ tively closely arranged. A few such groups have been indica¬ ted in Figs. 3a and 3b. There are relatively few blood ves- sels in the area adjacent to the catheter which indicates that IGF-II has no effect on the angiogenesis . In comparison IGF-I results in some proliferation of desmin-positive cells, but said cells are more scattered and do not tend to arrange in bundles in the same manner as after treatment with IGF-II (Figs. 4a and 4b). It is also seen that whereas IGF-II initiates selective proliferation of desmin- -positive cells, IGF-I, in addition to weak proliferation of such cells, activates also other cell components, as fibro- blaεts and blood vessels. This observation is a clear indica¬ tion that IGF-II, contrary to IGF-I, is useful in the regene- ration of muscle tissue at a site of damage. Thus, it can be seen that the treatment with IGF-II results in a well diffe¬ rentiated and structured muscle tissue thus eliminating the scar tissue that normally is irreversibly formed. Model for local treatment of heart muscle injury in rats. Male Sprague-Dawley rats, weighing about 350 g are used. They are allowed free access to food and water through¬ out the experiments. Muscle injury. Muscle injury is induced by intramuscular injection of Marcain. Marcain injection results in a selective damage to muscle cells, while the blood vessels are spared (Butcher 1989). The animals are anaesthetized with penthobarbital, 45 mg/kg b.w. intraperitoneally. An abdominal midline incision is made and, by penetrating the diaphragm, 0.25 ml of Marcain (2.5 mg/ml) is injected into the apical part of the myocar¬ dium. The midline incision is then closed and the animals are allowed to recover. Treatment of the injury. Drugs to be investigated are infused into the peri¬ cardium through a catheter connected to an osmotic -pump (alzet, model 2ML 1, pumping 7 days; or model 2ML 2, pumping 14 days). The catheter is inserted into the pericardium by a modified Seldinger technique. At a preselected time after induction of the heart muscle injury the animals are again anaesthetized with pen- thobarbital, 45 mg/kg b.w. , and the midline incision is re- -opened. A Venflon catheter (1.7 mm OD) is inserted a short distance into the pericardium, through the diaphragm, making sure not to damage the heart wall with the needle. The steel needle is then withdrawn, leaving the plastic catheter in place. A silastic tubing (0.025 in. ID; 0.047 in. OD) is in- serted through the catheter into the pericardium and finally, the Venflon catheter is withdrawn leaving the silastic tubing in the pericardial sac. The silastic tubing is secured to the abdominal wall by 6/0 silk sutures and is then connected to the osmotic pump in the peritoneum. After suturing the inci- sion the animals are allowed to recover. At the end of the experiment, after one or two weeks depending on the pump used, the animals are anaesthetized. The correct position of the silastic tubing is controlled by injecting a small volume of methylene blue through it. The animals are fixed by transcardial perfusion with 4% buffered ormaldehyde, taking care not to interfere with the area in¬ jured by the Marcain injection. After post- ixation for a further 3 hours the hearts are transferred to phosphate-buf¬ fered saline containing 7 . 5% sucrose, and are kept at +4°C until further processing. Histology . The progression of the healing process is assessed by immunostainings for intermediate filaments and acting iso- forms and by light microscopy on plastic sections. The apical part of each heart is divided by a sagittal section through the injured area. One part is embedded into methacrylate plastic (His'toresin, Reichert- ung) and cut into i μm thick sections. From the other part serial cryostat sec- tions are prepared and stained with monoclonal antibodies against des in (Dakopat), vimentin (Dakopat). The immunoreac- tion is visualized by a secondary HRP-linked antibody (Amers- ha ) followed by the DAB reaction. EXAMPLE 3 Following the procedure outlined above 5 μg of IGF-II per day are administered for 7 days. This IGF-II treatment is initiated 3 days after injection of Marcain. 0.15. BSA is used as a control. Treatment with BSA normally results in the for¬ mation of scar tissue with connected tissue and proliferating fibroblasts and granulation vessels. The result of the IGF-II treatment is illustrated in Fig. 5 a-d. Fig. 5a and 5b illustrate control and IGF-II treatment, respectively, using staining with monoclonal anti¬ bodies against des in. The white area enclosing the arrow indicates the damaged area in the cardiac muscle section, and it can be seen from comparison between said area of 5a and of 5b that a significant regeneration and healing has taken place due to the IGF-II treatment. Fig. 5c and 5d show a corresponding comparison between control and IGF-II treated sample using vimentin for the staining, and comparison between 5a and 5b reflects an essen¬ tial improvement in the healing process by the IGF-II treat¬ ment . EXAMPLE 4 Example 3 is repeated but the IGF-II dose is doubled to lOμg per day. The result of the IGF-II treatment is re¬ flected by an astonishing enlargement of the rat cardiac muscle compared to the control. Histological investigation does not indicate any connective tissue proliferation or da¬ mages which can be observed in secondary hypertrophy in con¬ nection with hypertonia. This unexpected result is a clear indication of usefulness of IGF-II treatment for the recovery of cardiac muscle capacity upon cardiac infarct. One particular indication where the present invention will be very useful is in the restoration of the performance on heart failure, i.e. coronary infarct. Many, if not all, of coronary infarct patients have to bear with an impaired coro- nary function in view of the formation of scar tissue in the cardiac muscle. Such scar tissue lacks the contracting/pump¬ ing function of normal cardiac muscle tissue and will thus result in reduced cardiac capacity. The present invention will also be useful in the - treatment of other smooth muscle tissue, such as vessel walls and intestinal walls, where healing and tissue regeneration will be positively affected.

Claims: CLAIMS 1. The use of. IGF-II for preparing a medicament for the regeneration of muscle tissue or the suppression of scar formation. 2. The use according to claim 1 for preparing a medi¬ cament for use in the healing of cardiac muscle tissue. 3. The use according to claim 1 for preparing a medi¬ cament for use in the healing of smooth muscle tissue. 4. A method for medical treatment for the purpose of regenerating muscle tissue, comprising the step of administe¬ ring to a patient in need of such treatment an effective amount of IGF-II. 5. A method according to claim 4 for the healing of cardiac muscle tissue. 6. A method according to claim 5 for the healing of smooth muscle tissue. 7. A method according to claim 4, 5 or 6, comprising the step of administering IGF-II topically at the site of defective tissue. 8. A method for minimizing scar formation during heal¬ ing of a wound, comprising the step of administering to a wounded patient an effective amount of IGF-II. 9. A method according to claim 8 for preventing unde- sired tissue adhesion after injury. 10. A method according to claim 9 for preventing unde- sired tissue adhesion upon surgery.

Inventors: JENNISCHE EVA, LAKE MATS, JENNISCHE, EVA, LAKE, MATS
Applicants: KABI PHARMACIA AB
Application year: 1992
Main classification: A61K37
IPC codes: A61K38, A61P43