Use of K-252a derivatives for enhancing the function of a stratial neuron

Abstract: Derivatives of K-252a of formulas (II), (III) and (IV) are useful for treating diseased neuronal cells.

Description: Background of the Invention Protein kinases are a broad class of enzymes which act to modify chemically many cellular proteins, by phosphorylation of amino acids. Inhibitors of protein kinases are structurally varied, and have variable (and sometimes contradictory) effects on the nervous system and other tissues. A given protein kinase inhibitor may influence more than one protein kinase. For example, K-252a, an alkaloid-like material isolated from the culture broth of Nocardiopsis sp. and Actinomadula sp. was originally reported to be a protein kinase C inhibitor, but was subsequently found also to inhibit protein kinases A and G, myosin light-chain kinase, and trk (a tyrosine kinase activated by nerve growth factor [NGF], the latter a neurotrophic protein which promotes the survival of peripheral, sensory and sympathetic neurons). Consistent with this latter effect, K-252a blocks the neurotrophic actions of NGF on PC-12 cells (chromaffin cells from rat adrenal medullary tumors, pheochromocytomas), and promotes the survival of dorsal root ganglion neurons and hippocampal neurons. However, it has been found to be cytotoxic at a wide range of concentrations, leading some investigators to conclude that it has limited usefulness in vivo . A microbial alkaloid related to K-252a, staurosporine, also has a variety of effects on different protein kinases and cell types. Staurosporine was found to have NGF-like effects on PC-12 cells, and to protect the gerbil hippocampus from post-ischemic injury. It is able to reverse damage to cholinergic neurons in the rat basal forebrain. K-252a and staurosporine have been proposed as tumor inhibitors. Staurosporine has been offered as an insecticide. Derivatives of staurosporine, with a hydrocarbyl or acyl substituent at the methylamine nitrogen, have been made and proposed for tumour inhibition, inflammation inhibition, immunomodulation, and treatment of diseases of the cardiovascular and central nervous systems. Nabeshima et al , The Journal and Exp. Therapeutics 257 :562-6 (1991), disclose that the use of staurosporine partially reverse reduced choline acetyltransferase activity. Summary of the Invention According to one aspect of the present invention, a functional derivative of K-252a is used for the manufacture of a medicament for use in enhancing, in a mammal, the function of a striatal neuron. The functional derivatives used in this invention are the respective compounds, of formulae II, III and IV, defined in claim 1. Description of the invention The invention provides a means for enhancing the function of striatal neurons, by administering to a mammal, e.g. a human, a therapeutic amount of one of the K-252a derivatives. The therapy may be given in conjunction with a neurotrophic factor, preferably a member of the neurotrophic family, and most preferably nerve growth factor (NGF). The neurotrophic family is a group of proteins with significant homology to NGF and includes, in addition to NGF, brain-derived neurotrophic factor (BDNF; Leibrock et al, Nature 341 :149-152, 1989); neutrophil-3 (NT-3; Hohn et al , Nature 344 :339-341, 1990); and neurotrophic-5 (NT-5; Berkemeier et al , Neuron 7 :857-866, 1991). By "enhancing the function of striatal neurons" is meant promoting striatal nerve cell survival, and/or nerve fibre (e.g. axonal) growth, and/or enhancing striatal activity of nerve cells. The therapy may involve the treatment where striatal neurons are injured, compromised, undergoing axonal degeneration, or at risk of dying. The compounds used in this invention can be formulated into pharmaceutical compositions by admixture with pharmaceutically-acceptable, non-toxic excipients and carriers. Such compounds are useful for administration to humans or other mammals who suffer from neurological diseases or disturbances characterised by increased risk of neuronal cell death or dysfunction. These neurological diseases and disturbances include but are not limited to: Alzheimer's disease; motor neuron disease including amyotrophic lateral sclerosis; Parkinson's disease; stroke or other ischemic injuries; Huntingdon's disease; AIDS dementia; epilepsy; concussive or penetrating injuries of the brain or spinal cord; and peripheral neuropathies. The functional derivative of K-252a used in this invention may possess improved solubility, absorption, transport (e.g. through the blood-brain barrier and cellular membranes), biological halflife, etc. Alternatively, or in addition, some modifications may decrease the toxicity of the molecule, or eliminate or attenuate any undesirable side-effect of the molecule. The 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 for use intranasally, particularly in the form of powders, nasal drops or aerosols. The composition may conveniently be administered in unit dosage form and may be prepared by any of the methods well known in the pharmaceutical art, for example, as described in 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, biocompatible, biodegradable lactide polymer, lactide/glycolide copolymer or polyoxyethylenepolyoxypropylene copolymers may be useful excipients to control the release of the active compounds. Other potentially useful parenteral delivery systems for these active compounds include ethylene-vinyl acetate copolymer particles, osmotic pumps, implantable infusion systems, and liposomes. Formulations for inhalation administration contain as excipients, for example, lactose, or may be aqueous solutions containing, for example, polyoxyethylene-9-lauryl 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 useful in 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., other growth factors which could facilitate neuronal survival or axonal growth in neurological diseases or disorders, for example, peripheral neuropathy. The concentrations of the compounds described herein in a therapeutic composition will vary depending upon a number of factors, including the dosage of the drug to be administered, the chemical characteristics (e.g., hydrophobicity) of the compounds employed, and the route of administration. In general terms, the compounds useful in this invention may be provided 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 µg/kg to about 1 g/kg of body weight per day; a preferred dose range is from 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. The functional derivatives of K-252a used in the invention may be prepared de novo by chemical synthesis using methods known to those skilled in the art. For example, procedures used for preparation of compounds of formula II are described in US-A-4923986. Procedures used for preparation of compounds of formula III are described by Moody et al , J. Org. Chem. 57:2105-2114 (1992); Steglich et al , Angew. Chem. Int. Ed. Engl. 19:459-460 (1980); Nakanishi et al , J. Antibiotics 39:1066-1071 (1986); and Japanese Patent Application No. 60-295172 (1985). Further methods are described for compounds II-1, 9, 12 and 15 in Japanese Patent Application No. 60-295173 (1985); for compounds II-2, 3, 4, 24, 25 and 26 in Japanese Patent Application No. 62-327858 (1987); for compound II-20 in Japanese Patent Application No. 62-327859 (1987); for compound II-10 in Japanese Patent Application No. 60-257652 (1985); and for compounds II-4, II-14, II-49, II-38, II-39, II-51, II-56, IV-2 and II-45 in European Patent No. 0651754. The following Examples illustrate the usefulness of compounds for use in this invention. Example 1 K-252a was assessed for its ability to promote survival in striatal cell cultures. Cell survival was measured by uptake of calcein AM, ana analog of the viable dye, fluorescein diacetate. Calcein is taken up by viable cells and cleaved intracellularly to flourescent salts which are retained by intact membranes of viable cells. Microscopic counts of viable neurons correlate directly with relative fluorescence values obtained with a fluorometric viability assay. This method thus provides a reliable and quantitative measurement of cell survival in the total cell population of a given culture. Striata were dissected from embryonic 17 day rat embryos and cells were dissociated by Dispase (neutral protease, Collaborative Research). Neurons were seeded at 5 x 10 4 /well (1.5 x 10 5 /cm 2 ) in 96-well plates onto wells previously coated with poly-1-ornithine and laminin. Cells were cultured in serum-free N2 medium containing 0.05% bovine serum albumin (Bottenstein and Sato, 1979) at 37°C in a humidified atmosphere, 5% CO 2 /95% air. Striatal neuronal survival was enhanced by K-252a in a concentration dependant manner. Maximum activity was found with 75nM K-252a, and produced an efficacy of 3-4 fold over the control (Figure 1). In the control cultures, 90% of the neurons plated on day 0 died within 5 days, whereas in cultures treated with K-252a, 50% of the neurons survived (Figure 2). The survival effect in striatal neurons occured after 3 days in culture and was sustained for at least 7 days in culture. These results are from a single application of K-252a on the day of culture inititation indicating a sustained effect of survival on a certain population of neurons. Figure 3 is a pair of photomicrographs taken from control cultures of cultures treated with 75 nM K-252a. There was an increase in cell survival and neurite outgrowth in these cultures in the presence of 75nM K-252a. Example 2 Figure 4 shows the effect of HBCS on kainate-induced neuronal degeneration in the hippocampus. Cannulated female Sprague-Dawley rats received 0.8 µg of HBCS, or vehicle, 40 minutes prior to and about 4 hours following kainate (0.6 µg) by icv infusion. Two weeks later the brains were excised, frozen, sectioned and stained for histological analysis, as described below. Data shown are the mean number of sub-regions of the hippocampus damaged for each group, ± SEM. HBCS significantly reduced the number of damaged areas within the hippocampus from about 2.5 ± 0.6 (without HBCS) to 1.3 ± 0.5 (with HBCS treatment). The effect of K-252a or its derivatives on kainate-induced neuronal damage was evaluated as follows : Adult male or female Sprague-Dawley rates (175-250 g) were anaesthetised with Nembutal (50 mg/kg, ip) and administered a drug, or vehicle treatment, in a total of 5 µl, before and after kainate treatment (5 µl) by icv infusion using a dose and infusion schedule as indicated above. Control animals received vehicle instead of kainate and drug infusion. For anatomical studies, icv infusions were delivered through a cannula (Plastic One, Roanoke, VA) implanted approximately one week before drug infusions, and positioned a sterotaxic coordinates: anterior-posterior at bregma, 1.5 mm lateral to bregma, and 4.4 mm ventral from the top of the skull. Results of this treatment were evaluated two weeks later using the anatomical analysis described below. In studies to assess the effect of K-252a or its derivatives on kainate-induced spectrin proteolysis, anaesthetised rats received a 5 µl infusion of the drug, or vehicle, simultaneously with kainate, through a 10 µl Hamilton syringe positioned at the sterotaxic coordinates described above. These rats were killed 24 hours later and subjected to biochemical analysis as described below. Anatomical analysis was performed as follows. Rats were killed by decapitation 2 weeks following treatments, and the brains rapidly removed and frozen on dry ice. A series of slide-mounted coronal sections from each brain was stained with thionin and examined microscopically. Damage to the hippocampus was quantified by summing the total number of 4 anatomically defined regions of the hippocampus (CA1-4 according to the classification of Lorente de No, as described by Shepard, 1979, The Synaptic Organisation of the Brain , Oxford, p. 310), on both left and right sides of the brain, that suffered a loss of pyramidal cells. Biochemical analysis was performed as follows. Calpain I-sensitive proteolysis of brain spectrin (fodrin) was evaluated in homogenates of the hippocampus using an immunoblot analysis described by Siman et al. (1988, Neuron, 1: 279-287). Briefly, rats were killed by decapitation 24 hours following treatment, and the dorsal hippocampus was rapidly dissected out of the brain and homogenized in 20 mM Tris-HCl (pH 7.4) containing 0.1 mM phenylmethylsulphonyl fluoride. Proteins from aliquots of each homogenate were separated by SDS-PAGE, and an immunoblot analysis was used to quantitate the amount of kainate-induced spectrin breakdown in each sample. Example 3 Thiry-one functional derivatives of K-252a were tested to determine their potency and efficacy in a striatal cell survival assay of Example 2. The following table shows data on 18 K-252a derivatives that promoted the survival of striatal neurons. Compound striatal survival % K-252a 25nM 75nM 100nM K-252a 100 III-1 69 NT - II-1 61 NT 104 II-35 52 81 - II-20 58 - NT II-10 91 - NT II-28 - 90 - II-5 104 NT - II-29 64 NT - II-2 65 - NT II-3 50 104 - II-30 - 114 NT II-6 81 NT - II-31 92 - NT II-32 69 NT - IV-3 - NT 47 IV-1 - NT 66 II-33 - 82 NT II-34 118 NT NT = not tested at that concentration - = not active at that concentration The following illustrates the effect of K-252a and functional derivatives thereof in the spinal cord ChAT assay, to determine their relative efficacy. Choline acetyltransferase (ChAT) activity was assayed in dissociated spinal cord cultures prepared from fetal rats by standard methods (see below). ChAT is the enzyme that catalyses the synthesis of the neurotransmitter acetylcholine, and is a specific biochemical marker for cholinergic neurons. In the spinal cord, the large majority of cholinergic neurons are motor neurons. Assay of this enzyme may thus be used as an indication of the effects of a factor (or factors) on the survival of cholinergic neurons and/or regulation of this enzyme. Experiments with dissociated cultures of fetal rat spinal cord cells were performed generally as described (Smith et al, J. Cell Biol. 101:1608-1621, 1985). Dissociated cells were prepared from spinal cords dissected from day 14 embryonic rats by standard techniques known to those skilled in the art, using trypsin dissociation of tissue (Smith et al, 1985). Cells were seeded (plated) at 6 x 10 5 cells/cm 2 in poly-1-ornithine coated plastic tissue culture wells in serum-free N2 medium and incubated at 37°C in a humidified atmosphere of 5% CO 2 /95% air (Bottenstein and Sato, PNAS USA 76: 514-517, 1979) for 48 hours. ChAT activity was measured using modifications of the Fonnum procedure (J. Neurochem. 24: 407-409, 1975) according to Ishida and Deguchi (J. Neurosci. 3:1818-1823, 1983) and McManaman et al, Dev. Biol. 125:311-320 (1988). Activity was normalised to total protein measured by the bicinchonicic acid/Cu ++ reaction (BCA protein assay reagent, Pierce, Rockland, Illinois, USA). Figure 1 shows that several compounds defined herein increased ChAT activity at 300 nM. Compound II-21, was also active at 30 nM (30% enhancement of ChAT activity over basal levels). This compound was more potent than K-252a or the remaining analogues since none of these actively enhanced ChAT activity at 30 nM. The following Table shows various compounds on ChAT activity in rat spinal cord cultures. Compound Spinal Cord ChAT Activity 300nM 30nM K-252a 100 - II-44 146 80 II-34 133 66 II-47 126 - II-1 122 - II-31 120 - II-29 118 65 II-32 118 70 II-38 118 - II-2 111 NT II-3 109 NT II-46 109 84 II-5 104 NT II-41 103 - II-42 102 64 II-4 100 - II-30 94 71 II-7 90 NT II-39 90 - II-36 86 - II-8 85 - II-9 83 64 II-10 81 143 IV-3 77 - II-11 76 NT II-21 76 63 III-1 75 - II-12 75 - IV-1 74 72 II-13 73 - II-14 71 - II-15 69 - II-18 68 - II-16 68 - II-40 66 - II-19 66 - II-20 65 65 II-45 65 - Compound Spinal Cord ChAT Activity 300nM 30nM II-22 62 - IV-2 62 NT III-2 60 NT II-25 58 - II-23 55 - II-24 50 - II-26 39 - II-33 - 77 II-43 - 125 II-49 - 62 II-51 130 53 II-56 88 53 II-48 70 - NT = not tested at that concentration - = not active at that concentration

Claims: Use of a compound for the manufacture of a medicament for use in enhancing, in a mammal, the function of a striatal neuron, the compound being a functional derivative of K-252a, represented by the formula wherein the following substitutions are made: Z 1 Compound R 1 R 2 X R Z 2 II-1 H H CH 2 N 3 OH H II-2 NHCONHC 5 H 5 H CO 2 CH 3 OH H II-3 CH 2 SOC 2 H 5 H CO 2 CH 3 OH H II-4 H H CH 2 OH OCH 3 H II-5 H H CONHC 2 H 5 OH H II-7 ( , ) H H CH 2 NH-Gly OH H II-8 H H CON(CH 3 ) 2 OH H II-9 H H -CH 2 NHCO 2 - H II-10 Br H CO 2 CH 3 OH H II-11 H H CONH 2 OH H II-12 H H CH 2 OH OH H III-1 -- H -- -- H II-13 H H CONHC 3 H 7 OH H II-14 ( , ) H H CH 2 NH-Ser OH H II-15 H H CH 2 SOCH 3 OH H II-16 H H CH=NOH OH H II-18 ( , ) H H CH 2 NH-Pro OH H II-19 H H CH-NNHC(=NH)NH 2 OH H II-20 Br Br CO 2 CH 3 OH O II-21 H H CONH(CH 2 ) 2 OH OH H II-22 H H CO 2 CH 3 OH O III-2 -- H -- -- O II-23 H H H OH H II-24 H H CH=NNHCONH 2 OH H II-25 H H CH 2 OCOCH 3 OH H II-26 H H -CH 2 OC(CH 3 ) 2 O- H II-29 NHCONHC 2 H 5 H CO 2 CH 3 OH H II-30 CH 2 SC 2 H 5 H CO 2 CH 3 OH H II-31 Br H CH 2 OH OH H II-32 Br Br CO 2 CH 3 OH H II-33 CH 2 SC 6 H 5 H CO 2 CH 3 OH H II-34 Cl Cl CO 2 CH 3 OH H II-36 H H CONHC 6 H 5 OH H II-37 H H CH 2 SO OH H II-38 H H CH 2 NHCO 2 C 6 H 5 OH H II-39 NHCONHC 2 H 5 NHCONHC 2 H 5 CO 2 CH 3 OH H II-40 N(CH 3 ) 2 H CO 2 CH 3 OH H II-41 CH 3 H CO 2 CH 3 OH H II-42 CH 2 OCONHC 2 H 5 H CO 2 CH 3 OH H II-43 NHCO 2 CH 3 H CO 2 CH 3 OH H II-44 Br Br CH 2 OH OH H II-45 Br Br CONHC 6 H 5 OH H II-46 Br Br CONHCH 2 CH 2 OH OH H II-47 CH 2 OC 2 H 5 H CO 2 CH 3 OH H II-48 CH 2 N(CH 3 ) 2 H CO 2 CH 3 OH H II-49 CH 2 SO 2 C 2 H 5 H CO 2 CH 3 OH H II-50 CH 2 S H CO 2 CH 3 OH H II-51 CH 2 SC 2 H 5 CH 2 SC 2 H 5 CO 2 CH 3 OH H II-52 CH=NNH H CO 2 CH 3 OH H II-53 CH 2 S H CO 2 CH 3 OH H II-54 CH 2 S(O) H CO 2 CH 3 OH H II-55 CH 2 S(O) H CO 2 CH 3 OH H II-56 CH 2 SC 2 H 5 CH 2 OH CO 2 CH 3 OH H II-57 H H CH 2 NHCO 2 CH 3 OH H II-58 Br H CONH 2 OH H II-59 H H CH 2 SC 6 H 5 OH H II-60 H H CH 2 S OH H II-61 H H CH 2 SOC 6 H 5 OH H IV-1 ( , ) H H -- -- H IV-2 Br H -- -- H IV-3 H H -- -- H IV-4 ( , ) H H -- -- H Use according to claim 1, wherein the compound is selected from II-1, II-2, II-3, II-4, II-5, II-8, II-9, II-10, II-11, II-12, II-13, II-15, II-19, II-20, II-21, II-23, II-24, II-25, II-30, II-32, III-1 and III-2. Use according to claim 1 or claim 2, wherein the medicament additionally comprises a neurotrophic factor. Use according to claim 3, wherein the neurotrophic factor is a member of the neurotrophin family. Use according to claim 4, wherein said member is nerve growth factor (NGF). Use according to any preceding claim, for use in the treatment of Huntingdon's disease.

Inventors: GLICKSMAN MARCIE A, KAUER JAMES C, LEWIS MICHAEL E, MATSUDA YUZURU, MURAKATA CHIKARA, NEFF NICOLA, ROBERTS-LEWIS JILL, SAITO HIROMITSU, GLICKSMAN, MARCIE A., KAUER, JAMES C., LEWIS, MICHAEL E., MATSUDA, YUZURU, MURAKATA, CHIKARA, NEFF, NICOLA, ROBERTS-LEWIS, JILL, SAITO, HIROMITSU
Applicants: CEPHALON INC, KYOWA HAKKO KOGYO KK, CEPHALON, INC., KYOWA HAKKO KOGYO CO., LTD.
Application year: 1993
Main classification: C07D487
IPC codes: A61K31, C07D519, C07D498, C07D487