GB2337702A - The use of staurosporine analogues for enhancing neurotrophin activity - Google Patents

Abstract

The staurosporine analogues BE-13793C, a monosaccharide derivative thereof, rebeccamycin and NB-506 are indicated for new therapeutic uses in conditions of neuronal degeneration such as Alzheimer's, Huntington's corea, epilepsy, and brain or spinal cord injuries. The compounds are believed to potertrate neurotrophin-3 (NT-3) action without unhibiting tyrosine kinase (Trk).

Description

- 1 2337702 THE USE OF STAUROSPORINE ANALOGUES FOR ENHANCING NEUROTROPHIN

ACTIVITY The present invention relates to the use of compounds which are staurosporine analogues to enhance molecular, biological and cellular activities which result from the binding of neurotrophins to cells which express neurotrophin receptors.

In particular the compounds potentiate neurotrophin-3 (NT-3) in embryonic rat dorsal root ganglia neurons without inhibiting tyrosine kinase (Trk) receptor signalling. Further they potentiate the action of NT 3 on Trk A autophosphorylation (activation) in Chinese hamster ovary (CHO) cells expressing human Trk A receptor and the activation of Trk A in rat PC 12 cells. They also potentlate the action of NT-3 on ALAP- kinase, one of the main downstream cell signalling steps activated by Trk A phosphorylation.

Related compounds are K252a (see US-A-5516772) and K252b (see EP A-675125) which are both derivatives of staurosporine. However the compounds used in the present invention do not inhibit Trk A activation at micromolar concentrations, unlike K252b which shows a toxic inhibitory effect in this range. Further the present compounds show greater effect on Trk A activation than K252b.

The present compounds have further benefits over staurosporine, K252a and K252b as they, do not totally inhibit protein kinase C activity in a cell free assay at concentrations up to 1OgM.

Background to the action of NT-3 and Trk receptors is known and is described, for example, in US-A-5516772.

Accordingly the present invention provides the use of a compound of formula:

H HN 0 1 0 N 0 N N H OH OH OH OH OH OH H 0 N 0 N N H OH OH OH OH C C) OH OH H 0 N 0 1 N H OH (B) or (D) N H OH H 0 N 0 N N H cl OH cl 011 OH Ome or a pharmaceutically acceptable salt thereof, for the manufacture of a medicament for enhancing a neurotrophin induced activity of a neurotrophin responsive cell.

There is also provided the use of a compound as defined above, or a pharmaceutically acceptable salt thereof, and an exogenous neurotrophin for the manufacture of a medicament for simultaneous, separate or sequential application for enhancing, a neurotrophin induced activity, of a t) neurotrophin responsive cell.

The neurotrophin is preferably NT-3.

The non-saccharide substituted compound (B) is known as BE-13793C 1,15 and is specifically, disclosed as an antitumour agent in EP-A-388956.

The dichloride derivative (D) is disclosed in WO-A-9604293 also only as an antitumour agent.

The aldehyde derivative (A) is specifically, disclosed as an antitumour agent in EP-A-545195. There is no suggestion whatsoever that the compound could also act to enhance the binding of neurotrophins to cells.

The monosaccharide derivative of BE-13793C (C) is disclosed in WOA9118003, again only as an antitumour agent.

As used herein the term 'enhancing' means that the combination of a compound of the present invention and a neurotrophin have a comparably greater effect on the induction of an activity than the neurotrophin alone.

As used herein, the phrase 'neurotrophin induced activity' means any response which directly or indirectly results from the binding of a neurotrophin to a neurotrophin responsive cell and which results in the autophosphorylation of ne urotrophin-recep tor associated tyrosine kinase. Most preferably, the neurotrophin receptor is a Trk receptor. Exemplary responses which directly or indirectly result from autophosphorylation of neurotrophin-receptor associated tyrosine residues are (1) choline acetyltransfe rase (ChAT) activity; (2) dorsal root ganglion (DRG) neurite outgrowth; (3) cell division (mitogenesis); or (4) promotion of the survival or function of cholinergic neurons and sensory neurons. These exemplary responses are implicated in the mediation and/or treatment of certain disorders, including (a) Alzheimer's, (b) motor neuron diseases (e.g., ALS, Parkinson's), (c) cerebrovascular disorders (e.g., stroke, ischaemia), (d) Huntington's, (e) AIDS dementia, (f) epilepsy, (g) peripheral neuropathies (e.g., those affecting DRG neurons in chemotherapy- associated peripheral neuropathy), (h) disorders induced by excitatory amino acids, as well as disorders associated with concussive or penetrating injuries of the brain or spinal cord. As such, the compounds of the present invention can be utilized in the medication andlor treatment, of disorders which result from, e.g., the death or dysfunction of cells to which a neurotrophin can bind, e.g., cholinergic or sensory neurons.

As used in the phrase 'neurotrophin induced activity' the term neurotrophin' includes both endogenous and exogenous neurotrophin, where 'endogenous' refers to a neurotrophin already present and exogenous' refers to a neurotrophin added to the system. As defined, neurotrophin induced activity,' Includes activity induced by: (1) endogenous neurotrophin; (2) exogenous neurotrophin; and (3) a combination of endogenous and exogenous neurotrophins.

As used herein, the phrase 'neurotrophin responsive cell' means a cell which expresses a receptor to which a neurotrophin can specifically bind.

Most preferably, the receptor is a Trk receptor. Exemplary neurotrophin responsive cells include neurons and non-neuronal cells.

As used herein, the term 'Trk' refers to the family of high affinity neurotrophin receptors presently comprising Trk A, Trk B and Trk C, to which a neurotrophin can bind and which binding leads to autophosphorylation of a tyrosine residue associated with such a membrane associated protein, and the direct or indirect activation of a functional response.

As used herein, the term 'neurotrophin' means a polypeptide that directly or indirectly promotes the survival or function of a cell, such as a neuron. Exemplary neurotrophins include Nerve Growth Factor (NGF), Neurotrophin-3 (NT-3), Neurotrophin-4/5 (NTA/5) and Brain Derived Neurotrophic Factor (BDNF).

Most preferably the Trk receptor is a Trk A receptor.

Tharmaceutically acceptable salts', as defined herein, are inorganic acid addition salts such as hydrochloride, sulfate, and phosphate; and organic acid addition salts such as acetate, maleate, fumarate, tartrate, and citrate. Examples of the pharmaceutically acceptable metal salts are alkali metal salts such as sodium salt and potassium salt, alkaline earth metal salts such as magnesium salt and calcium salt, aluminium salt, and zinc salt. Examples of the pharmaccutically acceptable ammonium salts are ammonium salt and tetramethyl ammonium salt. Examples of the pharmaceutically acceptable organic amine addition salts are salts with morpholine and piperidine. Examples of the pharmaceutically acceptable amino acid addition salts are salts with lysine, glycine and phenylalanine.

The compounds disclosed herein find utility, in a variety of settings. For example, in a research environment, the compounds can be utilized to investigate, refine and determine models for'downstream' effects of autophosphorylation as well as in elucidating the functional activities of the neurotrophins. Autophosphorylation of tyrosine residues of receptor linked tyrosine kinases (e.g., in Trk) is an absolute requirement for the activation of signal transduction pathways which regulate functional responses of, e.g., neurons; accordingly,, the disclosed compounds which, e.g., enhance such autophosphorylation, can be used in the development of in vitro assays for analysis of molecular mechanisms impacted by such autophosphorylation. In this way the disclosed compounds can be utilized 1,15 in the design of improved in vitro models for molecular mechanisms mediated by. Trk receptor binding to neurotrophins. The utility of the disclosed compounds in the design of model systems for the discover37 of neurotrophin- like agents is further underscored b3, the following: (1) the exact mechanism of the neurotrophin signalling pathway is not fully, understood; and (2) the association of neurotrophins with trophic and survival-promoting actions of neurons is also not fully understood.

Therefore, the present compounds can be used, e.g., in the discovery of agents which have marginal neurotrophin like activity in that such agents, when combined with the disclosed compounds, can be screened for enhancement of neurotrophin-induced activity.

Degeneration, death or non-functioning of neurons which result in nerve cell degeneration is a feature of many human neurological disorders, including, but not limited to, Alzheimer's; motor neuron disorders (e.g-, ALS, Parkinson's); cerebrovascular disorders (e.g., stroke, ischaemia); Huntington's; AIDS dementia; epilepsy; concussix7e or penetrating injuries of the brain or spinal cord., peripheral neuropathies (e.g., those affecting sensory neurons in che motherapy- associated peripheral neuropathy); and disorders induced by excitatory amino acids. Because the disclosed compounds are useful in enhancing neurotrophin induced activities of neurotrophin responsive cells (e.g., cholinergic, sensory or DRG neurons), the disclosed compounds beneficially lend themselves to utility as therapeutic agents. Thus, because the disclosed compounds have evidenced utility in, e.g., enhancement of ChAT activity or DRG neuron survival, the utility of the compounds in the treatment of disorders associated with, e.g., decreased ChAT activity or the death of DRG neurons, is within the scope of this disclosure.

The compounds disclosed in the present invention are preferably used to treat peripheral sensory neuropathies.

The compounds provided herein can be formulated into pharmaceutical compositions by admixture with p harm ace utically acceptable non-toxic exciplents and carriers. As noted above, such compositions may be prepared for use in parenteral administration, particularly in the form of liquid solutions or suspensions; for oral administration, particularly in the form of tablets or capsules; or intranasally, particularly in the form of powders, nasal drops, or aerosols.

The compositions may be conveniently administered in unit dosage form and may be prepared by any of the methods well known in the pharmaceutical art. See, Remington's Pharmaceutical Sciences (Mack Pub. Co., Easton, Pa., 1980). Formulations for parenteral administration may contain as common excipients sterile water or saline, polyalkylene glycols such as polyethylene glycol, oils of vegetable origin, hydrogenated naphthalenes and the like. In particular, blocompatible, biodegradable lactide polymer, lactide/glycolide copolymer, or polyoxyethylene polyoxvpropylc,ne copolymers may be useful excipients to control the release of the present compounds. Other potentially useful parenteral delivery Systems include ethylene-vinyl acetate copolymer particles, osmotic pumps, implantable infusion systems, and llposomes.

Formulations for inhalation administration contain as excipients, for example, lactose, or may be aqueous solutions containing, for example, polyoxyethylene-9-1auryl ether, glycocholate and deoxycholate, or oily solutions for administration in the form of nasal drops, or as a gel to be applied intranasally. Formulations for parenteral administration may also include glycocholate for buccal administration, methoxysalicylate for rectal administration, or citric acid for vaginal administration.

The materials for this invention can be employed as the sole active agent in a pharmaceutical or can be used in combination with other active ingredients, e.g., neurotrophins, or other factors (i.e., growth factors) or drugs which could facilitate neuronal survival or axonal growth in neurological diseases.

The concentration of a compound described herein in a therapeutic composition will vary depending upon a number of factors, including the dosage of the compound to be administered, the chemical characteristics (e.g., hydrophobicity) of the compounds employed, and the route of administration. In general terms, the compounds of this invention may be I 'ded 1 6 )rovl in an aqueous physiological buffer solution containing about 0.1% to 10% w/v compound for parenteral administration. Typical dose ranges are from about 1 pg/kg to about 1 g/kg of body weight per day; a preferred dose range is about 0.01 mg/kg to 100 mg/kg of body weight per day. The preferred dosage of drug to be administered is likely to depend on such variables as the type and extent of progression of the neurological disease, the overall health status of the particular patient, the relative biological efficacy of the compound selected, the formulation of the compound excipients, and its route of administration.

There is also disclosed a method for enhancing a neurotrophin induced activity of a neurotrophin responsive cell, such as Trk A in a patient requiring such enhanced activity, which method comprises administering to said patient a compound of formula:

H HN.L 0 1 (A) 0 N 0 N N H OH OH OH OH OH OH H 0 N 0 or N N H OH OH OH OH (C) OH OH or a p harm ace utically acceptable salt thereof.

H 0 N 0 1.

N N H H (B) OH OH (D) H 0 N 0 N N H cl OH cl OH OH ONIC Preferably the method comprises the simultaneous, sequential or separate administration of an exogenous neurotrophin, such as NT-3.

The compounds used in the present invention are made as described in EP-A-545195 (see Example 5), WO-A-9118003, WO-A-9604293, EP-A- 388956 and WO-A-9729205.

The following Methods illustrates the present invention:

METHODS (I) ELISA for dissociated dorsal root ganglia (DRGs) t, 15) DRGs were dissected and removed from either E16 rat embryos or Irlestation chick embryos, dissociated using try-psin and cultured das t, 1 ' -1 -gonto poly-D-lysine/laminin coated 96-well plates in Ham's F14 medium supplemented with either SATO (for 30 minutes with rat DRGs) or 10% foetal calf serum (two hours for chick DRGs). The medium was then aspirated and the cultures treated with compounds made up in F14 supplemented with SATO (final concentration: 4.3mg/ml BSA, 0.77gg/ml progesterone, 20Vtg/nil putrescine, 0.49iglml L-thyroxine, 0.048 gglml selenium and 0.42gglml tri-iodo-thyronine) for 48 hours at 370C 15% C02. Cultures were then fixed with 4% p araform aldehyde, treated with blocking serum (5% normal horse serum in P13S/0.3% TX100) for one hour, followed by incubation with a primary mouse monoclonal antibody, raised to GAP-43 (L500 in blocking serum, Sigma) overnight. Cultures were then treated with a peroxidase conjugated sheep anti-mouse secondary antibody (L1000 in blocking serum, Sigma) for one hour. K-blue, a peroxidase substrate was then added in order to visualise the reaction (blue colour change); the intensity of colour obtained being indicative of GAP-43 im munore activity. In order to quantify increases in GAP-43 imm unore activity, the optical densities of plates were then read at 650nin.

For each plate, a standard curve to NGF was incorporated and data obtained for all compounds expressed as a percentage of NGF control.

b In order to visualise the DRGs for the purpose of cell counting, a third layer blotinylated rabbit anti-sheep antibody (L200, Vector) was added to the wells for 30 minutes, followed by incubation with a peroxidase conjugated avidin-blotin complex for 30 minutes. Cells were then visualised using a peroxidase substrate (Vector SG) and counted by eye.

(11) Image analysis with explanted ganglia Primary cultures of chick dorsal root ganglion explants were prepared by dissecting DRGs from 10 day gestation chick embryos and t, Y plating them into 3-dimensional collagen gels before incubating for a period of two hours at 370C / 5% C02 to allow gels to set. Compounds were then added in Ham's F14 supplemented with SATO, and the cultures returned to the incubator for 48 hours. Following this, the cultures were then fixed with 4% p araform aldehyde and placed in blocking serum (10% normal horse serum in PBS11% TX100) overnight. Thereafter, explants were incubated with a mouse monoclonal antibody to GAP-43 (L500 in blocking serum) overnight followed by incubation with a blotinylated horse anti-mouse secondary antibody (L500 in blocking serum) overnight. A peroxidase conjugated avidin-blotin complex was then added overnight to the cultures and staining visualised using Vector SG peroxidase substrate.

Explants were then mounted onto glass slides. Neurite outgrowth in these cultures was quantified using MCID image analysis to measure the amount of pixels occupied by neurites; the area of the whole DRG was established using densitometry, from which the area occupied by the body of the DRG was subtracted. Data are expressed as the mean s.e.m.

(111) Western blotting b CHO cells expressing human TrkA or rat PC 12 cells were serum deprived for two hours and then left unstimulated or stimulated with either compound and/or NT-3 for ten minutes. The cells were lysed in RIPA buffer and TrkA was immunoprecipitated using the agarose conjugated anti-Trk antibody SC-139 (20il). The resulting immunoprecipitated pellets were then electrophoresed on a 6% Tris glycine gel and probed by western blotting using the antiphosphotyrosine antibody 4G10 (O.Ipl/ml) and a secondary anti-mouse-HRP conjugate (Amersham; 1/1000 dilution). The phosphorylated bands were then visualised by ECL.

Results Figure 1 shows typical standard curve showing NGF-induced elevation of GAP-43 immunoreactivity in primary cultures of rat dissociated dorsal root ganglia. Results are expressed as percentage of control group (n=4 per treatment group, data expressed as mean s.c.m).

b 1,15 This graph illustrates that DRGs of this type are NGF-dependent for survival, and that, the ELISA method described can detect compounds acting through a TrkA type mechanism.

Figure 2 shows the effect of K252b and Compound (A) on GAP-43. immunoreactivity in primary cultures of rat dissociated dorsal root ganglia, as shown by an ELISA. Results are expressed as percentage of NGF control group (n=4 per treatment group, data expressed as mean s.e.m). Neither Compound (A) or K252b show any increase in GAP-43 im m unore activity at the concentration range depicted in the figure, suggesting a lack of a survival effect in the absence of added NT-3 in this assay.

Figure 3 shows the effect of K252b and Compound (A) in combination with NT-3 on survival of rat dissociated DRGs using a GAP 43 ELISA. Mean data are expressed as percentage of NGF control s.e.m (n=4 per treatment group per experiment). Results show that both K252b and Compound (A), at nanomolar concentrations, potentiate the modest increase in GAP-43 immunore activity achieved with NT-3 alone. However, at micromolar concentrations, K252b causes a sharp decline in immunore activity in contrast to the plateau effect seen with Compound (A). Two replica experiments have been performed providing similar results.

The following table sets out the results of this experiment:

Table 1 treat K252b Compound (A) Y SEM Y SEM Control 117.83 12.89 76.09 4.16 NT-3 lOng/ml 143.91 28.93 106.52 11.03 10iiM + NT-3 230.00 32.32 112.17 13.40 10Onl\1 + NT-3 307.83 47.87 122.17 15.03 50OnM + NT-3 325.22 49.51 172.17 5.14 luM + NT-3 207.83 14.98 161.30 17.25 5uM + NT-3 61.30 6.40 197.39 35.16 1OuM + NT-3 62,17 4.40 185.65 14.71 Table 2 gives cell counts from an ELISA plate of rat dissociated DRG cultures, showing the effect of Compound (A) and K252b (50OnM) on NT- 3-induced elevation of GAP-43 immunoreactivity. Data confirms the results obtained using the GAP-43 ELISA, whereby both K252b and Compound (A) potentiate survival effects of NT-3 at nanomolar concentrations, but only Compound (A) maintains this survival effect at micromolar concentrations.

TABLE 2

Control NGF NT-3 K252b (50OnM) Compound (A) (50OnM) (10Ong/m1) (1Ong/inl) + NT-3 + NT-3 (1Ong/nil) (10nglml) 3033 152 1309 567 21 2632 94 911 424 19 2857 123 938 429 18 2960 117 774 425 2871 122 983 461 1 87 12 114 35 Mean SEM Figure 4 gives ELISA data showing the effect of Compound (A) in either the presence or absence of NT-3, on primary cultures of dissociated chick DRGs. Results are expressed as percentage of NGF control, mean s.e.m (ii=4 per treatment group). Results indicate that Compound (A) does not possess any neurotrophic activity itself, but cause., a marked potentiation of the moderate trophic effect seen with NT-3.

Figure 5 shows neurite out-rowth in primary cultures of chick DRG explants in response to K252b and Compound (A). Data are expressed as the mean of three DRGs per treatment group s.e.m. Results indicate a lack of a neurotrophic effect on such explants with either compound at nanomolar concentrations.

Figure 6 gives effects of Compound (A) and K252b on NT-3-induced neurite extension in primary chick DRG explants. Results for K252b plus NT-3 are expressed as the mean of 4 DRG explants per treatment group (except for 10OnM K252b + NT-3 where n=3) s.e.m- results for Compound (A) plus NT-3 are expressed as the mean of three separate experiments s.e.m. Data show that both Compound (A) and K252b potentiate the neurotrophic effect obtained in the presence of NT-3, but this is small in comparison to that seen with explants treated with NGF.

Figure 7 shows photomicro graphs of 10 day incubation chick DRG explants treated with NT-3, a combination of NT-3 and Compound (A) or NGF. Explants were grown in collagen gels (Guthrie & Lumsden, 1994) and treated for 48 hours as follows: (A) control, (B) NT-3, lOng/ml (C) 10OriM Compound (A) + lOnglml NT-3(D) 50OnM Compound (A) + lOnglml NT-3, (E) lOng/ml NGF. The photographs show that there is no outgrowth in control cultures, moderate outgrowth in NT-3 treated ganglia and an increase in the amount of outgrowth in ganglia treated with a combination of Compound (A) and NT-3.

Figure 8 shows a Western blot analysis of TrkA autophosphorylation in different cell lines. CHO cells expressing human Trk-A (A) or rat PC-12 cells (B) were serum-starved for 2h and then left unstimulated or stimulated with the indicated concentration of compound and/or neurotrophin-3 (NT-3) for 10 min. The cells were lysed in RIPA buffer and TrkA was immunoprecipitated using the agaro se -conju gate d anti-Trk antibody SC-139 (20gL). The resulting immunoprecipitated pellets were then electrophoresed on a 6% Tris-glycine gel and probed by Western blotting using the antiphosphotyrosine antibody 4G10 (O.Ipg/mL) ty k> k, - 14 and a secondary antimouse-HRP conjugate (Amersham; 1/1000 dilution).

The phosphorylated bands were visualised by, ECL.

Claims (6)

1. The use of a compound of formula:

H HN 0 1 0 N 0 N N H OH OH OH (A) OH OH (B) OH 11 0 N 0 01, N N H OH OH OH OH (C) OH OH (D) H 0 N 0 1 N N H H OH OH H 0 N 0 N N H c] OH cl OH OH ON1e or a pharmaceutically acceptable salt thereof, for the manufacture of a medicament for enhancing a neurotrophin induced activity of a neurotrophin responsive cell.

2. The use of a compound as defined in claim 1, or a pharmaceutically acceptable salt thereof, and an exogenous neurotrophill b 1 15 for the manufacture of a medicament for simultaneous, separate or sequential application for enhancing a neurotrophin induced activity of a neurotrophin responsive cell.

3. The use defined in claim 2, wherein said exogenous t) neurotrophin is NT-3.

4. A method for enhancing a neurotrophin induced activity of a neurotrophin responsive cell, in a patient requiring such enhanced activity, which method comprises administering to said patient a compound of formula:

H HN 0 1 0 N N N H OH OH OH 011 (A) OH OH H 0 N 0 N N H OH OH OH 011 OH OH ( C) or a pharmaceutically acceptable salt thereof.

H 0 N 0 N N H H (B) OH OH H 0 N 0 T N H cl OH cl OH (D) 11 0Me

5. The method of claim I comprising the simultaneous, sequential or separate administration of an exogenous lie urot rop hill.

6.

is NT-3.

The method of claim 5 in which said exogenous neurotrophin