EP0548083A1

EP0548083A1 - Nerve-regenerating agent

Info

Publication number: EP0548083A1
Application number: EP19910911250
Authority: EP
Inventors: Peter Dr. Med. Wehling
Original assignee: Individual
Current assignee: Individual
Priority date: 1990-06-15
Filing date: 1991-06-17
Publication date: 1993-06-30
Also published as: DE4019208A1; AU7968491A; WO1991019508A1

Abstract

Une composition stimule la régénération des nerfs après un évènement traumatisant, tel que le sectionnement d'un nerf, ou lors de lésions dues à des brûlures ou à des rayonnements. A cet effet, on utilise les substances messagères du système immunitaire, de préférence des substances sélectionnées dans le groupe des kinines, notamment des cytokines et des lymphokines, des leucotriènes, des prostaglandines, des interleukines et des interférons, ainsi que des combinaisons de celles-ci.A composition stimulates the regeneration of nerves after a traumatic event, such as the cutting of a nerve, or during lesions due to burns or radiation. For this purpose, the messengers of the immune system are used, preferably substances selected from the group of kinins, in particular cytokines and lymphokines, leukotrienes, prostaglandins, interleukins and interferons, as well as combinations thereof. this.

Description

Abnormal regeneration means B E S C H R E I B U N G

The invention relates to the manufacture of a medicament for the regeneration of nerves, the function of which is impaired due to a traumatic event or otherwise, for example due to burns or radiation damage.

In mammals and also in humans, many more nerve cells initially develop than are later used in the development of the nervous system. So-called neurotrophic factors then decide in the further course of development which nerve cell remains alive and which does not. It can only survive if a nerve cell makes contact with a target tissue via its axons - the stimulating cell processes - in which the neurotrophic substance necessary for its growth is contained or is produced. If this contact, e.g. interrupted by a traumatic event, the cell dies.

The longest known neurotrophic factor is the peptide NGF (nerve growth factor). NGF keeps both sympathetic and some types of sensory cells alive. In addition to the nerve growth factor NGF, other proteins have recently been discovered that enable the survival of the respective nerve cells. For example, the peptide BDNF (brain-derived neurotrophic factor), which is primarily responsible for the functionality of sensory nerve cells and neurons that are involved in the visual process. As well as factors not specified in more detail, such as the fibroblast growth factor FGF, the ciliary neurotrophic factor CNTF, the purpurin and the activin, for which a survival effect has so far only been demonstrated in vitro, that is to say in cell culture. The starting point for the task was investigations by Lindholm et al. (Nature, Vol. 330, 658 ff (1987); J. Biol. Chem., Vol. 263, 16348 ff (1988)) on the intracellular regulation of NGF synthesis after its stimulation by interleukin-1. These experiments show, inter alia, that the addition of 11-1 in non-neuronal cells (fibroblasts) of the rat sciatic nerve leads to an increase in the intracellular level of the NGF messenger RNA. Immigrated macrophages that release 11-1 have the same effect in vivo. It is also known that fibroblasts have a high number of IL-1 receptors and that all non-neuronal cells of the sciatic nerve have the ability to NGF synthesis (Heumann et al. (1987) J. Cell Biol, Vol. 104, 1623 ff).

Despite all the scientific studies, there were but so far no drug that prevents the death of nerve cells and nerve cells after a traumatic event, such as a division, or cell regeneration Nerven¬ ^"and fibers promotes. Cytokines and lymphokines such as interleukins, prostaglandins , Leukotrienes, Interleukins, and also Interferons are humoral inflammation mediators, which have a tissue-destroying effect in so-called inflammatory diseases. Synovial cytokines were therefore considered to be nerve-toxic. The inflammation also increases the spontaneous activity of the nerve, which is important for the person concerned Patients are extremely painful, which is why it was assumed that these experiments, which were carried out essentially in vitro, were of no importance for medical practice.

The object of the invention is to provide a medicament which promotes the regeneration of nerves after a traumatic event, such as a severing of the nerve, or in the event of burns and radiation damage. This object is achieved according to claim 1 by the use of messenger substances from the immune system for the manufacture of a medicament for the regeneration of nerves.

The messenger substances are advantageously selected from the group of kinins, in particular cytokines and lyphokines, that is to say leukotrienes, prostaglandins, interleukins and interferons, and combinations thereof. Interleukin-1 is very particularly preferred.

At first glance, it may seem completely absurd to assume that kinins such as interleukin-1, which is mainly produced by macrophages and monocytes but also by other cells, have healing properties as well as tissue-destroying effects. This opposite effect can, however, be explained by the pathophysiology of the inflammation and the diverse properties and functions of the messenger substances of the immune system.

For example, interleukin has a variety of biological functions in the immune response: it activates the T cells by inducing the production of interleukin-2 and its receptor; it induces fever; it increases connective tissue absorption and stimulates fibroblasts and synovial cells (e.g. synovial cells HIG-82, synovial skin cells), which themselves also produce interleukin-1, for the release of protaglandins.

Interleukin contains two related proteins, the alpha and the beta form of interleukin 1, both of which react with the same receptor. However, due to this structural relationship, both interleukins should be particularly suitable for the production of a medicament for the regeneration of nerve cells. With regard to the treatment of people, the use of genetically derived, human inter - I _

leukine, which will shortly also be approved by the Federal Health Office.

The interleukin-1 is advantageously contained in the medicament between 50 and 5000 units / ml if the medicament is not subsequently diluted.

Another major clinical problem is scar formation in the vicinity of the severing site. The nerve severance leads to two competing pathophysiological processes at the severing site: on the one hand, the amputated neuron tries to regain its old extent by sprouting appendages in the distal part of the injury (cellular regeneration) - the number of However, nerve cells remain unchanged - on the other hand, this is countered by injury-related fibroplasia and scar formation, whereby the number of connective tissue cells increases. The new connective tissue cells then block the free space distal to the injury and thus the nerve regeneration.

This leads to the approach of changing the granulation tissue with the aim of improving the nerve regeneration result.

The process of wound healing can be inhibited at various levels. Preferred here is, among other things, the addition of connective tissue-dissolving and / or scar-destroying substances to the medicament: Such substances are, for example, collagenases and / or peptidases, such as chymopapain, papain, chymotrypsin, trypsin, etc. The addition of collagenase in a concentration between is very particularly preferred 100 - 10,000 U / ml. The addition of substances such as proline derivatives, in particular cis-hydroxyproline, is also advantageous 7 -

and / or D-pencillamine, which inhibit the synthesis of collagen and collagen fibers in vivo.

Thus, in the presence of cis-hydroxyproline, this is incorporated into the procollagen instead of proline, which results in reduced collagen formation. In the rat model, nerve regeneration - compared to a control nerve - actually shows an increase in the neural myelin sulfate with a simultaneous decrease in collagen by 47% in the distal segment. D-penicillamine can also weaken newly formed collagen in its structural stability.

The addition of hormones and factors which promote nerve growth and / or delay wound healing is also advantageous. Corticosteroids and their derivatives, progesterone, estrogen, methyl-prednisolone, triamzinolone acetate, as well as insulin, PDGF (platelet-derived growth factor), GH (growth hormone), FGF (fibroblast growth factor), CNTF (ciliary neurotrophic factor) are recommended for this ), Purple, activin, and descendants thereof. For example, steroids have an anti-inflammatory effect for weeks and suppress wound healing. Estrogen and progesterone also show a similar effect in wound healing. The topical application of triamzinolone acetate also shows an improvement in nerve regeneration.

For reasons of surgery and healing, it is further advantageous if the medicament additionally contains a fibrin glue made of aprotinin-CaCl∑; Thrombin and fibrinogen; Tissucol ^W2 (Red List: 47047). The medicament can additionally have surfactants, solvents, solubilizers, stabilizers, antioxidants, such as dithioerythritol, in order to ensure the durability and effectiveness of the composition. - c -

The form of administration of the medicament is advantageously adapted to the treatment method. Due to the different possible uses, the following forms of application of the drug appear to be advantageous: as an injection solution, as an infusion solution for local and / or systemic infusions, as a therapeutic system, e.g. as a dispenser implant, dispenser balls, as a solution and jelly for spreading or as an intermediate filling.

The advantageous effects of the medicament according to the invention in the regeneration of nerves result from various animal experiments. The results of the tests are shown graphically in FIGS. 1-5. Show it:

Figure 1 is a scheme for performing animal experiments;

FIG. 2 shows the clinical evaluation of the animal experiments with regard to the average motor power of the right hind paw;

FIG. 3 shows the representative derivation of the evoked spinal potential and the evoked muscle activity in the intrinsic foot muscles after irritation of the sciatic nerve proximal or distal to the severing point on a test animal, whereby in addition to the epidural suture and fibrin seal, the medicament according to the invention was used;

FIG. 4 analogously to FIG. 3, a representative derivation of the evoked spinal potential and the evoked muscle activity in the intrinsic foot muscles after irritation of the sciatic nerve, only one animal from the control group was used; FIG. 5 shows the change in the amplitude of the evoked spinal potential in the test and control group as a function of the time after the operation;

FIG. 6 shows the height of the amplitudes in the intrinsic foot muscles measured after stimulation of the sciatic nerve proximal to the nerve severing in the control and test group;

FIG. 7 shows the average values of the measured nerve conduction speed (NLG) in the control and experimental group; and

8a shows the absolute total number of fibers in the test and control group 5 mm distal to the nerve severing point 12 weeks after the severing and

Figure 8b their relative frequency as a function of the total fiber diameter of the myelinated nerve fibers.

Carrying out animal experiments

The surgical procedure was as follows: In nembutal anesthesia (40-50 mg / kg body weight), the right sciatic nerve of the Wistar rat was exposed laterally up to the branching in the tibial and peroneal nerves. Then 3 marker threads (9-0 Ethilon ^W2 , BV-4, monofilament, polyamide, from Ethicon) were placed epineurally in the longitudinal direction to prevent the nerves from rotating. The nerve was severed with a smooth cut about 1 cm proximal to branching with the aid of microscissors.

The epineural suture was made with 6 threads of 9-0 Ethilon. Approx. 1mm proximal and distal of the separation point between the nerve slowly injected with 0.05 ml of dissolved IL-beta (50 U; from Genzy e) at a concentration of 1000 U / ml. The injection was slow, so that the continuation of the 11-1 solution into the severing area could be followed under the surgical microscope. The rat interleukin-1 beta used had a molecular weight of 17,000 daltons; it was dissolved in buffer solution shortly before the injection. The seam area was then treated with 0.05 ml of fibrin glue (100 IU aprotinin calcium chloride / ml + 50 IU thrombin and fibrinogen; Tissucol ^TM) coated, in order to prevent escapes that the interleukin-1 from the tionsbereicht Transsek-. The experimental group ^" consisted of 5 animals.

The control group (6 animals) was treated and examined immediately. However, no interleukin-1 but physiological saline was injected.

The animals in the experimental and control group were observed over a period of 3 months. The animals were completely neurophysiologically examined before the operation as well as 7, 12, 15, 25, 32, 40, 60 (80th, for IL-1) and 90 days after the operation. In order to be able to rule out an autonomous denervation behavior (biting the hind paw) of the animals, they were photographed in the same interval. After 3 months, all test animals and 3 control animals were assessed "clinically" and πtorphometrically. The course of the experiment and the analyzes is shown schematically in FIG. 1.

Clinical findings

The clinical findings are shown graphically in FIG. 2. The SFI according to de Medinacelli is plotted in percent and the ordinate shows the postoperative time. The graphic shows the Average motor performance of the right hind paw in the experimental (11-1 + suture / fibrin) and control group (suture / fibrin only). Medianaceli shows standard values with standard deviation of the clinical index (SFI = sciatic functional index). The experimental group comprises 5 animals and the control group 6 animals. In the test group, the clinical motor performance is increased on average. The under- . difference is significant (p <0.05). In the experimental group, a weakened toe spread reflex was to be triggered in all animals after 90 days. In the control group, however, only 3 animals showed this reflex, the others not. ^'

The values of the test group are represented by oblique lines and dots. They are (mean) m = -79% ^' , (standard deviation) sd = -12.4. The values of the control group are shown by black bars. Mean m = -102%, standard deviation sd = -9.3. After 60 days, the values in the test group were m = -93%, sd = -11 and in the control group m = -106% sd = -11.3. No differences were found at earlier measuring points.

Neurophysiological findings

For the neurophysiological assessment of the thus atosensitive system, the evoked spinal potentials were carried out at the level L1 after simultaneous irritation of the tibial nerve distal to the severance at the level of the Achilles tendon insertion. To assess the motor system, the muscle response of the intrinsic foot muscles (CAMP) evoked by irritation of the N, sciatic proximal and distal to the cut was derived. A main focus was placed on the amplitude of the muscle response, which is used as a measure of motor reinnervation. - 1 C -

Figure 3 shows the representative derivative of _. evoked spinal potential (SSEP L1, left lane) and the evoked muscle activity in the intrinsic foot muscles (CAMP, right lane) after irritation of the proximal sciatic nerve (CAMP) or distal (SSEP) of the severed parts on a test animal e ^'iner epidural seam and Fibrinab¬ seal and using the invention Medika¬ management. The animal in question developed complete foot paresis immediately after severing the nerve, which improved significantly from the 60th clinical day. The filter setting was LF: 10 Hz + HF: 10 kHz, the SSEP (left lane) corresponds to 64 averaged answers. The CAMP (right lane) corresponds to a unique irritation of the nerve. Supramaximal stimuli were used. If it was not possible to determine the threshold value due to complete paresis, the irritation was caused by applying 40 mV.

Note the significant increase in amplitude in the CAMP after 60 and 90 days (Examples D and E), which was accompanied by a clear clinical improvement. The clinical and neurophysiological measurements of the non-operated opposite side were normal.

FIG. 4 shows an analogous investigation on a control animal. The severed parts were only treated by epidural suture and fibrin sealing, but no treatment with interleukin was carried out.

The control animal also developed a complete foot paresis immediately after severing the nerve, which only improved slightly over time. The filter setting was LF: 10 Hz + HF: 10 kHz. The SSEP on the left corresponds to 64 averaged answers. The CAMP corresponds to a unique irritation of the nerve. Supramaximal stimuli were used. Was a threshold setting not possible due to complete paresis, was stimulated with 40 mV.

In comparison to FIG. 3, note the slight increase in CAMP on the 60th and 90th postoperative day. The somatosensitive response differs from the test animal in that the SSEP occurs late. The clinical and neurophysiological measurements of the non-operated opposite side were normal.

Evoked spinal potential

FIG. 5 shows the amplitude values (SSEP-Ll) of the evoked spinal nerve potentials in μV at level L1 in the test and control group as a function of the time after the operation. The irritation occurs in the right tibial nerve distal to the nerve severance at the level of the Achilles tendon. The point of irritation was thus approx. 3.5 cm from the severing point.

On the 15th postoperative day there was a significant difference (p <0.001) between the two groups u. The associated measured values were:

Postop. Day trial control significance

0 25.0 µV p <0.923;

12 0, uV

15 9.833 µV p <0.001;

19 10.5 µV p <0.085;

25 10.5 µV p <0.22;

32 9.66 µV p <0.492

40 9.66 µV p <0.195

60 10.44 µV p <0.133

90 14.8 µV p <0.54)

Average values m are given in each case.

The first negative peak to determine the latency was not clear due to the curved curve be determined. Due to this methodological uncertainty, the calculation was therefore omitted. In contrast to the CAMP, the reproducible derivation of the amplitude of the SSEP in the regenerating nerve was also made more difficult in the course of the observations. This is also shown by an increased standard deviation compared to the CAMP experiments.

Evoked muscle activity of the intrinsic foot muscles

(CAMP)

The results are shown graphically in FIG. 6. The level of the amplitudes in the intrinsic foot muscles (in mV) measured after excitation of the sciatic nerve proximal to the nerve severance.

In the group treated with 11-1 (checks), a significant increase in the amplitude of the evoked muscle potential compared to the control group (black squares) can be observed after 60 and 90 days.

Measurement results (mean m) and comparison of experimental and control group preop .: 5 mV / 4.58 mV; sd: 0.5 / 1.29; 7th freedom; p <0.618.

40 days postop: m: 0.12 mV / OmV; sd: 0.179 / 0.9; 9. Freedom Gr. p <0.131.

60 days postop .: m: 1.04 mV / 0.133 mV; sd: 0.467 / 0.327; 9th freedom: p <0.004.

90 days postop .: m: 3.1 mV / 1.517 mV; sd: 0.477 / 0.436; 9th freedom; p <0.0004.

90 days postop .: m: 3.1 mV / 1.517 mV; sd: 0.447 / 0.436; 9th freedom; p <0.0001). The mean values with standard deviation correspond to derivatives of a total of 11 animals (test group n = 5; control group n = 6)

The evoked muscle response of the intrinsic foot muscles in the aforementioned experiment on the treated side showed a high level of reproducibility with an intra- and inter-individual constant increase in the amplitude of the CMAP to be observed in the two groups from the 40th to 60th postoperative day. Up to the 32nd postoperative day, no potential in the dependent foot muscles could be derived in both groups on the treated side. From the 40th postoperative day in the experimental group (11-1, 5 animals) a potential could be derived in 2 animals. In contrast, this was not possible with any of the animals in the control group.

While one of the control animals had a demonstrable potential on the 60th postoperative day, this was observed in all animals of the experimental group. At the end of the observation period, a CAMP could be triggered in all animals in both groups. The increase in the amplitude of the CAMP in both groups was largely associated with the postoperative observation period (this was not observed in the SSEP), in particular the intraindividual evaluation gave a continuous increase in the amplitude.

Motor nerve conduction speed (NLG)

FIG. 7 graphically shows and evaluates the height of the motor NLG in m / s (vertical axis) preoperatively and 90 days postoperatively in the test and control group. The average values with standard deviation of the measured nerve conduction velocity (NLG) are shown. The NLG was determined from the derivation of the muscle response after irritation of the sciatic nerve. ximal and distal of the nerve severing area. If a CAMP was not detectable at two different stimulation sites, this animal was not included in the static calculation of the NLG.

After 60 days, the NLG could be determined in one animal in the control group; in the test group (with interleukin-1), however, in all. After 90 days, the NLG could be measured in all animals of both groups. The difference in motor nerve conduction speed after 90 days of treatment is significant.

Morpho etrical investigation

The result of the morphometric examination in the test and control group is shown in FIGS. 8a and b.

They show that there is no difference between the experimental and control groups with regard to the relative distribution of the total fiber diameter. However, the absolute total number of fibers in the test and control group distal to the nerve transection site is significantly different. This confirms that more nerves actually grow back after treatment with interleukin

Figure 8a shows the total number of myelinated fibers 5 mm distal to the nerve severing site 12 weeks after the severing. The counting shows that in the group Versuchs¬ the total fiber number (m: 13336) is significantly higher relative to the Kon ^¬ roller (9685 m). This difference has a significance level of p <0.004. FIG. 8b shows the relative frequency (%) (vertical axis) as a function of the total diameter of yelinated fibers (μm) 3 months after the cuts (test group = weak bars; control group = hatched bars). The total nerve fiber diameter is indicated on the horizontal axis in steps of 0.5 μm. The mean diameter of the axon was 2.108 μm (sd: 1.032) in the test group and 2.271 μm in the control group (sd: 1.068). The G ratio was m: 0.539 (sd: =, 097) in the test group and m: =, 587 (sd: 0.095) in the control group. All other ^* parameters of the two groups were also the same.

The results show, compared to a control group, a significant improvement in neurophysiological parameters and clinically functional values, the total number of myelinated axons 5 mm distal to the transection was increased, the morphometric parameters remained unchanged.

The pathophysiological mechanism behind this improved functional nerve regeneration result can only be assessed hypothetically. The membrane properties of the nerve fibers are possibly stabilized in the sense of improved resistance to damaging influences which take place in the submicroscopic range.

Claims

PATENT CLAIMS

1. Use of messenger substances of the immune system for the manufacture of a medicament for the regeneration of nerves.

2. Use according to claim 1, characterized in that the messenger substances are selected from the group of kinins, in particular cytokines and lymphokines, leukotrienes, prostaglandins, interleukins and interferons, and combinations thereof.

3. Use according to claim 1 or 2, characterized gekenn¬ characterized in that the messenger is interleukin-1 (IL-1).

REPLACEMENT LEAF 08

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4. Use according to one of claims 1 to 3, characterized in that the messenger is alpha-interleukin-1 (IL-la).

5. Use according to one of claims 1 to 3, characterized in that the messenger is beta-interleukin-1 (II-Lß).

6. ^' Use according to any one of claims 1 to 5, characterized in that the beta-interleukin-1 between 50 - 5000 units / ml is contained in the medicament.

7. Use according to one of claims 1 to 6, characterized in that the messenger is genetically engineered human interleukin-1.

8. Use according to one of claims 1 to 7, characterized in that the medicament additionally comprises connective tissue-releasing and / or scar-destroying substances.

9. Use according to claim 8, characterized in that the medicament additionally has collagenases and / or peptides.

10. Use according to claim 8 or 9, characterized gekenn¬ characterized in that the medicament contains between 100 - 10,000 U / ml collagenase.

11. Use according to one of claims 1 to 11, characterized in that the medicament additionally has substances such as proline derivatives, in particular cis-hydroxyproline, D-penicillamine, which inhibit the synthesis of collagen and collagen fibers in vivo.

12. Use according to one of claims 1 to 10, characterized in that the medicament additionally growth-fσr- changing hormones and factors such as progesterone, estrogen, methyl-prednisolone, triamzinolone acetate, corticosteroids, insulin, PDGF (platelet-derived growth factor), GH (growth hormone), FGF (fibroblast growth factor), CNTF (ciliary neurotrophic factor) , Purpurin, Activin, and their derivatives.

13. Use according to one of claims 1 to 12, characterized in that the medicament additionally contains a fibrin glue made of aprotinin-CaCl2; Thrombin and fibrinogen; Tissucol ^wz (Red List: 47047).

14. Use according to one of claims 1 to 13, characterized in that the medicament additionally has surfactants, solvents, solution mediators, stabilizers, antioxidants, such as dithioerythritol.

15. Use according to one of claims 1 to 14, characterized in that the application form of the medicament is selected from the group consisting of injection solution, infusion solution for local and / or systemic infusion, therapeutic system, such as dispenser implant, dispenser balls, solution and jelly For painting.