CZ20032060A3 - Pharmaceutical preparation - Google Patents

Abstract

Tacrolimus derivatives having high levels of neurotrophic activity and low levels of immunosuppresive activity. These compounds are useful as neurotrophic agents, particularly, for preventing or treating neuronal injury/dysfunction.

Description

Pharmaceutical composition

Technical field

The invention relates to the use of the neurotrophic analogue tacrolimus for the manufacture of a pharmaceutical composition having a neurotrophic effect.

BACKGROUND OF THE INVENTION

Certain macrolide compounds such as tacrolimus and related agents may prevent or treat cerebral ischemia, such as have been described, for example, in WO 94/14443. It is known that some pipecolinic acid derivatives having affinity for FKBP-type immunophilin, for example tacrolimus, stimulate the growth of damaged peripheral nerves or promote neuronal regeneration according to WO 96/40140. Also, some non-immunosuppressive compounds, such as geldanamycin and analogs thereof, disrupt the steroid receptor complex and promote nerve growth according to WO 99/21552.

SUMMARY OF THE INVENTION

It has now been found that a certain tacrolimus analog, a compound of formula I, has an excellent neurotrophic effect, but unlike tacrolimus, it has little or no immunosuppressive effect. As will be further demonstrated, the compound of formula I has good neurotrophic activity compared to tacrolimus, for example measured by its ability to prolong neurite length. Similarly, it has been shown that administration of a compound of formula I can induce axon regeneration and rapid regeneration after nerve or spinal cord injury. Furthermore, the compound of formula I has these advantageous effects with little or no immunosuppressive activity compared to tacrolimus.

Accordingly, the present invention provides the use of a compound of formula I as a very good neurotrophic agent having substantially no immunosuppressive activity, in particular the use of a compound of formula I for the manufacture of a pharmaceutical composition having a neurotrophic effect.

Unexpectedly, it has been found that the compound of formula I can be used for the prevention or treatment of neurological dysfunctions or injuries caused by direct injury or disease of the nervous system, or at least ameliorating such conditions with little or no immunosuppressive effect .

Accordingly, the compound of formula I is useful for the treatment of damage or dysfunction, which conditions may be caused by physical injury, nutritional disorders, ischemia, degenerative diseases, malignancies, infectious diseases or by the action of drugs, toxins or poisons.

For example, the compound of formula I can improve the damage or dysfunction caused by surgical procedures on the nervous system, peripheral nerve injury, burns, encephalomyelitis, HIV, herpes virus, malignancies, radiation, drug interactions, folic acid deficiency or vitamin B12 deficiency or also by the action of neurotoxins or chemicals, for example lead.

Thus, the compound of formula I can be used for the manufacture of pharmaceutical compositions for the prevention or treatment of nerve damage and dysfunction such as polymyositis (multiple myositis), Guillan-Barré syndrome, Menier syndrome, polyneuritis (multiple neuritis), mononeuritis (single nerve inflammation) Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis (ALS), Huntington's disease, radiculopathy, neuropathy such as diabetic neuropathy or neuropathy induced by chemotherapy and the like, spinal cord injury, senile dementia, vascular dementia, multiple sclerosis and the like.

The compound of formula I has the following structure:

It can be prepared according to Example 29 of US 5376663. It will be understood that it may have one or more stereoisomers, both optical and geometric isomers relative to asymmetric carbon atoms and double bonds, all of which may be used in accordance with the present invention. with the essence of the invention.

A compound of formula I may form pharmaceutically acceptable salts, derivatives, solvates or precursors, all of which are included within the scope of the invention. Of the solvates, hydrates and ethanolate are usually most preferred.

The preferred form of the compound of formula I is represented by the structure

The compound of formula I may be administered as a pure substance or in admixture with other compounds or other ingredients, preferably a pharmaceutical carrier or carrier.

When used as a pharmaceutical, the compound of formula I may be mixed with an organic or inorganic carrier, vehicle or excipient suitable for topical administration, oral, enteral, subcutaneous, intravenous, intramuscular or parenteral administration. It may be a solid, semi-solid or liquid composition which contains, as active ingredient, a compound of formula (I) and, in addition, one or more carriers which may be carriers. • Environment or excipients. Typical excipients are conventional pharmaceutical carriers, other active ingredients, buffers, dispersants, emulsifiers and other excipients.

Thus, the compound of formula I may be formulated with non-toxic conventional pharmaceutically acceptable carriers into tablets, pellets, capsules, eye drops, suppositories, solutions, for example saline, emulsions, suspensions, for example, olive oil, ointments, aerosols, creams , patches and other dosage forms.

Suitable carriers include water, aqueous sodium chloride solution, dextrose solution, oils including animal, vegetable and synthetic oils and some petroleum products. Other suitable carriers may be, for example, glucose, lactose, acacia, gelatin, mannitol, starch paste, magnesium silicate, talc, corn starch, keratin, colloidal silica, potato starch, urea and other carriers suitable for the preparation of solid, semi-solid or liquid. resources.

It is also possible to use stabilizers, emulsifiers, thickeners, colorants, flavorings and perfumes.

The compound of formula I is used in a pharmaceutical composition in an amount effective to produce the desired effect on the disease process or condition. Preferably, the agent is used in an amount sufficient to produce a neurotrophic effect or to stimulate nerve cell growth.

Mammals which may be administered a pharmaceutical composition comprising a compound of Formula I include domestic

• ·· • · · · • · · · • · · · • · · · · · · · · · • · · · • · · · · • · · · · • · · · • · · · • · · ·

animals such as cows, horses, pigs, dogs, cats, rats, mice, rabbits, hamsters, and the like, can be primates as well as humans.

A preferred route of administration of a composition comprising a compound of Formula I is by injection or oral administration.

The effective dose of a compound of Formula I may vary from patient to patient and is dependent upon the age and general condition of the patient. The daily dose will usually be in the range of 0.0001 to 1000 mg, preferably 0.001 to 500 mg and in particular 0.01 to 100 mg, the average single dose will be in the range of 0.001 to 0.1 mg, 0.2 to 0 5 mg, usually 1 mg, 5 mg, 10 mg, 50 mg, 100 mg, 250 mg or 500 mg is administered. The daily dose for chronic administration in humans will be in the range of 0.1 to 30 mg / kg / day. The compound of formula I may also be administered concurrently or sequentially with other agents having a neurotrophic or nerve cell growth stimulating effect.

The pharmaceutical composition of the invention may be administered to mammals, for example, humans to induce neuronal regeneration and functional nerve regeneration and to stimulate neurite outgrowth in the treatment of various neuropathological conditions including injury to the peripheral nerves and the central nervous system caused by injury, for example spinal cord injury, sciatic injury. or facial nerve, limb transplantation after amputation, and the like, may also be administered to various nerve diseases such as diabetic neuropathy, conditions after cancer treatment, for example, neuropathy induced by acrylamide, taxol, vinca alkaloids and doxorubicin; it may be, for example, articulation disorders, turbid consciousness, disorders • 4 · 4 · 4 · 4 · 4 · · · · ·

4 4 4 4 4 4

444 444444 4 * 4 44 * 4 * 44

44 44 44 44 momentum such as associated with cerebral infarction, cerebral hemorrhage and the like and other neurological disorders, including various peripheral neuropathic and neurological disorders such as trigeminal neuralgia, glossopharyngeal neuralgia, Bell's disease, mysthenia gravis, muscular dystrophy, amyotrophic lateral sclerosis , progressive muscular atrophy, progressive congenital atrophy of the ocular muscles, ruptured or prolapsed intervertebral disc, cervical spondylosis, diseases of nerve knitting, neuropathy, especially peripheral nerves, caused for example by the action of lead, acrylamides, gamma-diketones, carbon monoxide, Barré, Alzheimer's disease, Parkinson's disease and Huntington's chorea.

Peripheral nerve disruption or spinal cord injury can be treated by administering a stimulating amount of a compound of Formula I and using a peripheral nerve graft, for example, an allogeneic graft according to Osawa et al., J. Neurocytol. 19, 833-849, 1990, Buttemeyer et al., Ann. Plastic Surgery 35, 396-401, 1995, or an artificial neural graft may be used according to Madison and Archibald, Exp. Neurol. 128, 266-275, 1994. Wells et al., Exp. Neurol. 146, 395-402, 1997. The space between the broken ends of the peripheral nerve or spinal cord is preferably filled with a special cell-free material such as colleague, methylcellulose and the like, or a cell suspension that promotes the growth of nerve cells. Xu et al., J. Neurocytol. 26, 1-16, 1997, olfactory cells and cover cells according to Li et al., Science 277, 2000-2002, 1997. The nerve growth promoting compound may be co-administered with such cellular or non-cellular padding material, or 44 44 44 4444

4 4444 44 4 • 44 «444 44 4

444 444445 4 4

4 44 44 4444

44444 44 44 49 44 can be administered systemically before, during or after grafting.

In particular, the compound of formula I may be used for the treatment or prevention of neural dysfunction, such as polymyositis (multiple myositis), Guillan-Barré syndrome, Meniere's syndrome, polyneuritis (multiple neuritis), mononeuritis (single nerve inflammation), Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis ALS, Huntington's disease, radiculopathy, neuropathy, for example diabetic neuropathy or neuropathy induced by chemotherapy and the like, spinal cord injury, senile dementia, vascular dementia, multiple sclerosis and the like.

The following examples are intended to illustrate the invention.

DETAILED DESCRIPTION OF THE INVENTION

Example 1

The compound of formula I statistically significantly prolongs the neurites of the hippocampal neurons

Preparation of cell cultures

Embryonic hippocampal neurons were obtained from rat fetuses at embryonic gout 18.5 (E18.5) according to Banker and Cowan, Brain Research, 1977, 126, 397-425. The hippocampal regions were removed, crushed and incubated in 100 I.U. Papain at 37 ° C for a total of 45 minutes, then the cells were resuspended in complete medium for neurons, which is the minimal baseline medium without the minimum baseline medium. Φ · · ♦ φφ φφ φφ · Φ

L-glutamine (GIBCO, Grand Island, NY) containing

1.5 ml / 100 ml High Glucose Minimal Medium (GIBCO), 0.1 ml / 100 ml Serum (Hito + Tm, Collaborative Research Inc., Lexington, ΜΑ), glutamine (GIBCO) and 5% fetal calf serum (GIBCO).

Cells were seeded onto coverslips at 500 cells per capped, coated with poly-L-lysine.

The slides were then mounted on single-layer pre-coated dishes of the cerebral cortex.

Axon length analysis of hippocampal neurons

Hippocampal neurons, identified by their characteristic polarity and dendrites, were monitored daily and randomly photographed for 72 hours (9-12 photos per cap). Axon, which is defined as the longest projection, was measured in photographs using a Houston HI-PAD connected to an IBM XT computer with appropriate Bioquant IV, R & M Biometrics software,

In Nashville, TN, only protuberances that were at least 3 times longer than the cell body were measured. Data from the same treated slides (3 or 4 per group) did not differ from each other and for this reason the data were combined. Mean values were calculated and compared (groups treated with compound Ia or tacrolimus were compared, control groups included), using the ANOVA program, followed by multiple comparisons according to Newman-Kulls (WINKS 4.62, professional edition).

Results • Φ «4

4 4 4 · · · · ·......

·· ··

44 · Φ ·· · 4 · · · ·

3 «· *

72ο 72 hours, no statistically significant difference was observed between the untreated control group and the 10 nM tacrolimus treated group. However, in the group treated with a concentration of 10 nM of the compound of formula Ia, there was a statistically significant neurite outgrowth as shown in Table 1.

Table 1. Effect of compound Ia and tacrolimus on mean neurite length in primary hippocampal cell cultures after 72 hours.

Neurite length (μιη) No treatment 372.3 ± 18.23 Tacrolimus 10 nM 423.4 ± 19.50 Compound Ia 10 nM 502.2 + 23.61 *

* P <0.5 vs untreated controls at the above statistical evaluation

Example 2

After treatment with Compound I on the average neurite length in SH-SY5Y human neuroblastoma cells

Preparation of SH-SY5Y neuroblastoma cell culture

SH-SY5Y human neuroblastoma cells were maintained in DMEM (Gibco) supplemented with 10% fetal calf serum (Sigma), 50 IU / ml penicillin and 50 µg / ml streptomycin (Gibco), and the cells were grown at 30 ° C in a 7% carbon dioxide. The cells were then seeded in 6-well plates at 15,000 cells / well and treated with 0.4 μΜ aphidicoline (Sigma). After five days, the cells were washed with a

nerve growth factor NGF at a dose of 10 ng / ml was added to them in the presence or absence of 10 nM tacrolimus or 1 nM compound Ia. After 96 hours, the medium was replaced with fresh medium for an additional 72 hours, for a total of cells incubated for 168 hours. In all experiments, duplicate embodiments were used and the average of the values for each group was calculated.

Neurite length analysis of neurobastastic cells SH-SY5Y

To analyze the length of the processes, the cells were randomly photographed after 168 hours (20 fields per well). Neurite length was measured in photographs using a Houston HI-PAD connected to an IBM XT computer equipped with appropriate software (Bioquant, IV, R&M Biometrics, Nashville, TN). Only those protuberances whose length was more than twice that of the cell body were measured. Data from the same wells were not different and were therefore combined. Mean values were calculated, which were then compared for the compound of formula Ia or tacrolimus and for samples treated with NGF alone. The ANOVA program was used, followed by the Newman-Kuels Comparative Test (Winks 4.62, Professional Edition).

Results

Neurite length measurements showed that both the compound of Formula Ia at 1 nM and tacrolimus at 10 nM statistically significantly increased neurite length after 168 hours compared to NGF alone at 10 ng / ml. However, the effect of 1 nM of the compound of formula Ia in combination with NGF was greater than that of 10 nM tacrolimus in combination with NGF.

Table 2. Effect of compound Ia and tacrolimus on the mean neurite length of SH-SY5Y human neuroblastoma cells after 168 hours.

Neurite length (μπι) No treatment 94.75 ± 3.734 NGF 10 ng / ml 198.8 + 8.991 Tacrolimus 10nM + NGF 10ng / ml 227.6 ± 9.130 * Compound Ia 1 nM + NGF 10 ng / ml 256.0 ± 9.067 * * P < 0.05 compared to MGF at svrc and described statistical

hodnoceni

Example 3

Compound I induces functional regeneration of the sciatic nerve in a rat after injury

Experimental animals and surgery of 6-week-old male Sprague-Dawley rats were anesthetized with 2% halothane, the right sciatic nerve was exposed and clamped 2 times for a total of 60 seconds in Dumont # 7 hip pliers. The wound site was marked with a sterile 9-0 suture, which was guided through the epineural cover.

Preparation of Compound Ia and Administration

Compound Ia was dissolved in a vehicle containing 30% DMSO and 70% saline. Three rats with injured nerve were given either Compound Ia at a dose of 1 or 5 mg / kg daily or an equivalent volume of vehicle at 5 ml / kg daily.

99 99

Evaluation of animal behavior

Animals were monitored daily up to 18 days.

The following scale was used to evaluate nerve function regeneration:

paralysis with limb turned out while walking, bent fingers ability to straighten limb and move fingers ability to walk on affected limb ability to stretch fingers while walking pedaling including heel and almost normal toe stretching

Animals whose ability was within the individual evaluation range were evaluated as follows: +, 0.25, ++, 0.5, +++, 0.75.

Tissue fixation and preparation

18 days after nerve injury, rats were deeply anesthetized with 4% halothane, heparinized and washed with 4% paraformaldehyde in 0.1 M sodium phosphate buffer pH 7.4 for a total of 10 seconds, after which the rats were washed with 1 liter of 5% glutaraldehyde in 0.1 M sodium phosphate buffer pH 7.4 and stored at 4 ° C for 24 hours. Tissue was harvested from the sciatic nerve at a distance of 5 mm from the injury site. In this study, only the data from the n.tibialis posterior arm supplying m.soleus were evaluated. The tissue was stored in 0.1 M sodium phosphate buffer pH 7.4, fixed for 2.5 hours with 1% osmium tetroxide in 0.1 M phosphate buffer, dehydrated in ethanol and embedded in plastic. Thin sections were stained with uranyl acetate and citrate

leaded, mounted on 75 mesh screens, placed on foil and monitored by JEOL 100 CX electron microscope.

Morphometric analysis

Axon size analysis was performed on n.soleus.

The number of regenerating myelinated axons was determined using an electron microscope. The mean values and the standard deviation were calculated for the vehicle-only group, the 1a / kg group of the compound Ia and the 5 mg / kg group for the same substance.

Statistical analysis

For behavioral analysis, mean values for function recovery were compared using ANOVA, followed by multiple comparisons by the Newman-Keuls test. In the case of morphometric analysis, the numbers of regenerated axons were compared using the same statistical methods.

Results

Functional regeneration

Functional regeneration was observed on days 15 to 17 and occurred earlier in rats treated with Compound Ia at 1 mg / kg and 5 mg / kg than in vehicle-only rats. The results are summarized in Table 3.

Table 3. Effect of compound Ia on functional regeneration of sciatic nerve in rats after injury.

Functional evaluation Carrier environment s.c. Compound Ia 30% DMSO v saline solution 1 mg / kg (s.c.) 5 mg / kg (s.c.) Day 15 1.67 ± 0.08 2.58 ± 0.17 * 3.17 ± 0.08 * Day 16 1.83 ± 0.08 2.83 ± 0.17 * 3.50 ± 0.00 * Den 17 2.50 + 0.00 3.08 + 0.22 * 3.50 ± 0.00 *

* P <0.05 using the aforementioned statistical evaluation

Electron microscopy

Morphological monitoring of the animals was performed 18 days after nerve injury.

The amount of regenerating myelinated axons per nerve area of 5000 pm ² was dramatically increased from 5.5 + 2.7 (mean + SEM) in vehicle-treated rats to 19 + 2.4 and 20 ± 2.9u rats, respectively. 1 and 5 mg / kg of Compound Ia were administered, P < 0.05. The results are shown in Table 4.

Table 4. Effect of compound Ia on the number of regenerating myelinated n.soleus axons 18 days after nerve injury in rats.

Carrier environment s.c. Compound Ia 30% DMSO v saline solution NaCl 1 mg / kg (s.c.) 5 mg / kg (s.c.) Regenerated myelinized axons 5.5 ± 2.7 19 ± 2.4 * 20 ± 2.9 *

* P <0.05 when comparing the active substance and vehicle to the above statistical methods ·· ····

Example 4

Compound I induces functional regeneration in a rat spinal cord injury model

1. Methods

Experimental animals and surgery of 6-week-old male Sprague-Dawley rats were anesthetized with 2% halothane, followed by laminectomy in the T10 / T11 region, and spinal cord in the same T10 / T11 region at half of its cross-section.

Preparation of Compound Ia and Administration

Compound Ia was dissolved in a vehicle consisting of 30% dimethylsulfoxide DMSO and 70% saline. Spinal cord injured rats were injected subcutaneously daily with 2 mg / kg of compound Ia or an equivalent volume of vehicle 5 ml / kg 7 weeks after surgery.

Evaluation of functional regeneration

Functional regeneration was evaluated according to a modified Tarlov / Klinger scale, walking on a narrow prism and a paw print test 2 weeks after injury.

A) Modified Tarlov / Klinger scale

The rats were left in the open for a period of time.

• w · · 4 · 4

minute and their movement was evaluated according to the following 0 to 6 scale:

damaged hind limb is barely detectable movement of damaged hind limb rapid movement in joints of hind limb, hips, knees or ankles without coordination, the limb does not hold any weight alternating steps forward, but the limb does not hold any weight limb keeps the weight walking with moderate difficulty in normal walking

B) Walking on a narrow prism

The rats were walked on wooden prisms 1.5 m long with decreasing width: 7.7 cm, 4.7 cm, 2.7 cm and 1.7 cm. The width of the narrowest prism was recorded, after which the rats were able to cross back and forth.

0 walk not possible i ia and none ( prism 1 walk is maybe on prism s width 7.7 cm 2 walk is maybe on prism s width 4.7 cm 3 walk is maybe on prism s width 2.7 cm 4 walk is maybe on prism s width 1.7 cm

C) Pawprint Test

The outer limbs of the rats were soaked in ink and the prints were monitored on paper that overlapped a narrow path of 60 cm long and 7.5 cm wide. Cases with at least 6 consecutive steps were evaluated, using the following scale:

· »·· ·· ···· ♦

9 9 9 9 9

9 999 9 9 «« · · · · · ·

The hind limbs touch the paper by the dorsal part, no footprint can be observed, at least three toes can be seen on at least three footprints foot rotation with more than twice the normal values can be observed, no stretching of the fingers can be observed, foot rotation occurs no inward or outward rotation observed, more than one heel footprint observed no heel footprint observed

Statistical analysis

As in the previous cases, the average values for each functional test were compared using the ANOVA program, followed by multiple Newman-Keuls comparisons.

2. Results

Functional regeneration

In all three functional recovery tests, impairment of motor function was improved using the Tarlov / Klinger evaluation (Table 5), prism walking (Table 6), and the paw print test (Table 7) following Compound Ia administration.

Table 5. Effect of Compound Ia on the Modified Tarlov / Klinger Assay in Spinal Cord Damage in Rats

0000

Modified rating Tarlov / Klinger Carrier environment s.c. 30% DMSO v saline solution NaCl Compound Ia 2 mg / kg (s.c.) Week 2 2.1 ± 0.1 3.7 ± 0.2 *

* P < 0.05 when comparing the active ingredient and vehicle environment using the statistical methods described above

Table 6. Effect of Compound Ia on Prism Walking in Prisms after Spinal Cord Damage.

Evaluation Carrier environment s.c. 30% DMSO v saline solution NaCl Compound Ia 2 mg / kg (s.c.) Week 2 0.9 ± 0.1 2.0 ± 0.3 *

* P < 0.05 when comparing the active ingredient and vehicle environment using the statistical methods described above

Table 7. Effect of Compound Ia on Footprint in Rat Spinal Cord Damage.

Evaluation Carrier environment s.c. 30% DMSO v saline solution NaCl Compound Ia 2 mg / kg (s.c.) Week 2 1.7 ± 0.4 3.6 ± 0.3 *

* P <0.05 when comparing the active substance with the vehicle as described above according to the statistical methods described above.

Φ · Φ · ····

Example 5

Compound Ia binds to FKBP12 but, unlike tacrolimus, has little or no immunosuppressive effect

1. FKBP12 binding assay

The binding assay was performed as described by Tamura K. et al., Biochemical and Biophysical Research Communications, Vol. 202, No. 1, 437-499, 1994. The results are summarized in Table 8.

2. MLR lymphocyte reaction

The MLR assay was performed according to US 4929611. The results are shown in Table 8.

Table 8. Pharmacological profile of compound Ia and tacrolimus in vitro.

IC ₅₀ for FKBP12 binding (nM) IC ₅₀ at MLR (nM) Compound Ia <5 > 100 Tacrolimus <5 <2

The above results demonstrate that Compound Ia does not have immunosuppressive activity, although it is capable of binding to FKBP12.

The results demonstrate a potent neurotrophic effect of the compound of formula I in both in vitro and in vivo models. In two cell culture models, the compound of formula I also increased at low levels.

99 concentrations of neurite outgrowth. In addition, systemic administration of this compound at low doses accelerated functional regeneration after nerve injury by increasing the rate of regeneration of sciatic nerve axons and also functional regeneration after spinal cord injury.

In addition, as shown above, the compound of formula I has a neurotrophic effect on stimulating nerve cell growth, although it does not have immunosuppressive activity. Thus, it is a valuable neurotrophic agent for stimulating or inducing the growth or regeneration of neurons, especially in cases where an immunosuppressive effect is not desired or inappropriate.

The invention also relates to a package comprising a packaging material and a compound of formula I stored therein, which substance is effective in preventing or treating neuronal dysfunction, the packaging material comprising a label indicating that the compound of formula I can be used to prevent or treating dysfunction or neuronal injury.

Said package is usually intended for commercial purposes.

The present invention also provides a pharmaceutical composition, such as a cell, tissue or graft suspension, which comprises cells that have been treated with a compound of formula I. Such a composition is useful for repairing damage to the nervous system. In addition to the active ingredient, it may contain other nerve cell growth stimulants, for example, other suspensions of those cell types that promote or assist the growth of nerve cells, such as myelin-producing cells, such as Schwann cells, oligodendrocytes, glial cells or cover cells. as well as extracellular matrix material such as collagen or others. 9 9999999 999 9 9 9 9

9 9 9 9 9 9 9 9 • 9 99 99 99 99 specific nerve cell regulators such as cytokines, mitogenic factors, immunophilins and neurotrophins, for example NGF-1, BDNF, CNTF, NT-3, NT-4 and NT-5 .

The grafts, for example homo-, allo- or xenomaterials, can also be treated with a compound of formula I to facilitate neuronal growth and use of these materials for transplantation and other applications.

It will be understood that many modifications and variations may be made within the scope of the invention, so that the invention may be put into practice other than as specifically described in the application. Many such modifications and variations will be apparent to those skilled in the art.

Thus, although the invention has been described in connection with specific preferred embodiments, it is to be understood that it cannot be limited to this embodiment and includes other embodiments as long as the necessary modifications are within the skill of the art.

Represented by:

//

Claims (42)

PATENTOVÉ Claims ·· ···· for the manufacture of a composition having a neurotrophic effect. The use of claim 1, wherein the composition is for preventing or treating neuronal dysfunction or injury. Use according to claim 1, wherein the composition is intended to stimulate or induce the growth or regeneration of nerve cells. Use according to claim 1, wherein the composition is intended to induce functional regeneration after nerve injury. Use according to claim 1, wherein the composition is for the prevention or treatment of nerve damage and dysfunction such as polymyositis (multiple myositis), Guillan-Barré syndrome, Menier's syndrome, polyneuritis (multiple neuritis), mononeuritis (inflammation of a single nerve) · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·· Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis ALS, Huntington's disease, radiculopathy, neuropathy such as diabetic neuropathy or neuropathy induced by chemotherapy and the like, spinal cord injury, senile dementia, vascular dementia, multiple sclerosis and the like. The use of claim 1, wherein the neuronal dysfunction or injury is Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, diabetic neuropathy, neuropathy induced by chemotherapy or spinal cord injury. Use according to claim 1, wherein the compound of formula I is in the form of a pharmaceutically acceptable salt, derivative, precursor or solvate. 8. A pharmaceutical composition comprising, as an active ingredient, a compound of formula I and ₃ % by weight of a compound of formula (I) and ₃ % by weight of a compound of formula (I). Pharmaceutical composition according to claim 8, characterized in that it comprises a compound of formula I in the form of a pharmaceutically acceptable salt, derivative, prodrug or solvate. Pharmaceutical composition according to claim 8, characterized in that the compound of formula I is used in the form represented by structure Ia. The pharmaceutical composition of claim 8, further comprising at least one additional neurotrophic agent. 12. A package or assembly comprising a wrapping material and a compound of formula I, wherein the wrapping material includes a label indicating that the compound of formula I is for preventing, ameliorating, or treating nerve dysfunction or injury. 13. A commercial package or assembly comprising a wrapping material and a compound of formula I, wherein the wrapping material includes a label indicating that the compound of formula I is for preventing, ameliorating, or treating nerve dysfunction or injury. 14. A process for the production of a neurotrophic pharmaceutical composition, which comprises admixing a compound of formula I with a pharmaceutically acceptable carrier, vehicle or excipient. 15. A method of preventing, ameliorating, or treating neuronal dysfunction or injury, comprising administering an effective amount of a compound of Formula I. • 4 ···· • 44 • 4 4 4 • 4 4 4 4 4 43 4 · «4 44 44 4 4 4 4 4 4 4 4 4 4 4 4 4 4 44 44 16. The method of claim 15, wherein the subject to be treated is a mammal. 17. The method of claim 15, wherein the subject to be treated is a human. The method of claim 15, wherein the composition is for preventing or treating nerve damage and dysfunction such as polymyositis (multiple myositis), Guillan-Barré syndrome, Menier's syndrome, polyneuritis (multiple neuritis), mononeuritis (multiple myositis). single nerve inflammation), Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis (ALS), Huntington's disease, radiculopathy, neuropathy such as diabetic neuropathy or neuropathy induced by chemotherapy and the like, spinal cord injury, senile dementia, vascular dementia, and multiple sclerosis. 19. The method of claim 15 wherein the neuronal dysfunction or injury is Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, diabetic neuropathy, neuropathy induced by chemotherapy or spinal cord injury. 20. A method of stimulating or inducing growth or regeneration of nerve cells, comprising contacting nerve cells with a compound of formula I. 21. A method for increasing the growth or regeneration rate of axons or nerve cells, comprising contacting nerve cells with a compound of formula I. fcfc fcfc fcfc fcfc fcfc fcfc fcfc fcfc fcfc fcfc fcfc 22. A method of increasing the rate of growth of nerve cells in a tissue, comprising administering to the tissue a compound of Formula I. 23. The method of claim 22, which is brain tissue, spinal cord tissue, or peripheral nerve. 24. A method of inducing functional regeneration after nerve injury, comprising administering an effective amount of a compound of Formula I. 25. The method of claim 24, wherein the nerve injury is a burn, traumatic or mechanical injury, surgery injury, physiological injury, pathological injury or immunological injury. 26. A method of treating a disrupted peripheral nerve or spinal cord, comprising contacting the damaged nerve or spinal cord endings with an effective amount of a compound of Formula I. 27. A method of treating a disrupted peripheral nerve or spinal cord, comprising administering a stimulating amount of a compound of Formula I and depositing a graft in the peripheral nerve or spinal cord. 28. The method of claim 27, wherein the method is an allograft. * • • •. *....... 9 · 29. The method of claim 27, wherein the method is an artificial neural graft. The method of claim 27, further comprising filling the space between the interrupted ends of the peripheral nerve or spinal cord with a non-cellular padding material. The method of claim 27, wherein the space between the broken ends of the peripheral nerve or spinal cord is filled with a cell suspension. 32. A composition comprising nerve cells treated with Compound I. 33. A tissue comprising nerve cells treated with Compound I. 34. A graft comprising nerve cells treated with compound I. 35. The graft of claim 34, wherein the graft is a homo-, allo- or xenograft. 36. A method of inducing functional regeneration after nerve injury, comprising administering an effective amount of a composition comprising cells treated with Compound I. 37. The method of claim 36, wherein the nerve injury is a burn, traumatic or mechanical injury, a surgical injury, or a trauma. physiological damage, pathological damage or immunological damage. 38. A method of treating a disrupted peripheral nerve or spinal cord, comprising contacting the ends of a damaged peripheral nerve or spinal cord with an effective amount of a composition comprising cells treated with Compound I. 39. A composition comprising cells, tissues or grafts treated with a compound of formula I and at least one neuronal cell growth promoting agent. 40. The composition of claim 39, wherein the nerve cell growth promoter is selected from the group of cell suspensions, nerve growth promoter, non-cellular material to fill the slit, or nerve growth factor. 41. The composition of claim 39, wherein the nerve growth promoter is collagen or methylcellulose. 42. The composition of claim 39, wherein the nerve growth promoter is a cell suspension. Represented by: