Compositions and methods for potentiation of neurotrophin activity

Compositions and methods for use in modulating neurotrophin activity, wherein the active agent is at least one compound which potentiates neurotrophin activity. A preferred class of active agents is K-252 compounds, including both microbial metabolites and derivatives thereof. Neurotropin activity is modulated by administration of an effective amount of at least one compound which potentiates neurotrophin activity. Potentiation of NT-3 by K-252b, K-252a, KT5720, and KT5823 provides a model for therapeutic intervention in a variety of neuropathological conditions.

BACKGROUND OF THE INVENTION
 The present invention relates to compositions which are useful in
 potentiating neurotrophin activity, as well as methods for the preparation
 and use thereof.
 Protein growth factors of the neurotrophin family, which includes nerve
 growth factor (NGF), brain-derived neurotrophic factor (BDNF),
 neurotrophin-3 (NT-3), neurotrophin-4 (NT-4) and neurotrophin-5 (NT-5)
 regulate nervous system development [Barde, Y-A., "Trophic factors and
 neuronal survival," Neuron 2:1525-1534 (1989); Thoenen, H., "The changing
 scene of neurotrophic factors," Trends Neurosci 14:165-170 (1991);
 Leibrock, J. et al., "Molecular cloning and expression of brain-derived
 neurotrophic factor," Nature 341:149-152 (1989); Ernfors, P. et al.,
 "Identification of cells in rat brain and peripheral tissues expressing
 mRNA for members of the nerve growth factor family," Neuron 5:511-526
 (1990); Hohn, A. et al., "Identification and characterization of a novel
 member of the nerve growth factor/brain-derived neurotrophic factor
 family," Nature 344:339-341 (1990); Maisonpierre, P. C. et al.,
 "Neurotrophin-3: a neurotrophic factor related to NGF and BDNF," Science
 247:1446-1451 (1990); Rosenthal, A. et al., "Primary Structure and
 Biological Activity of a Novel Human Neurotrophic Factor," Neuron
 4:767-773 (1990); Jones, K. R. and Reichardt, L. F., "Molecular cloning of
 a human gene that is a member of the nerve growth factor family", Proc.
 Natl. Acad. Sci. USA 87:8060-8064 (1990); Hallbook, F. et al.,
 "Evolutionary studies on the nerve growth factor family reveal a novel
 member abundantly expressed in Xenopus ovary," Neuron 6:845-858 (1991);
 Berkemeier, L. R. et al., "Neurotrophin-5: a novel neurotrophic factor
 that activates trk and trkB," Neuron (in press)]. In addition, the
 neurotrophins are strongly implicated as playing an important role in
 structural maintenance, plasticity and repair of the adult nervous system
 [Hefti, F. et al., "Function of neurotrophic factors in the adult and
 aging brain and their possible use in the treatment of neurodegenerative
 diseases," Neurobiol. Aging 10:515-533 (1989)].
 Neurobiological research carried out in recent years has confirmed that
 development, maintenance of function and regeneration of neurons is
 profoundly influenced by the neurotrophic factors. These neurotrophins
 stimulate mechanisms necessary for survival, neurite growth and functions
 related to transmitter production and release. For example, it has long
 been known that nerve growth factor (NGF), the first and best
 characterized neurotrophin, is a neurotrophic factor for peripheral
 sympathetic and sensory neurons, and more recent findings show that NGF
 also affects cholinergic neurons in the brain. NGF is required by
 sympathetic and dorsal root ganglion cells for survival during embryonic
 and early postnatal life, and is also critical to the normal function of
 these neuronal types in adult animals. NGF is further implicated in the
 regulation of a variety of developmental processes such as
 naturally-occurring cell death, differentiation, process outgrowth and
 synaptic rearrangement.
 Experiments over the last few decades have yielded evidence that NGF
 regulates a variety of cellular processes important for neuronal function.
 Administration of pharmacological doses of NGF to rodents results in
 striking increases in ganglion cell size, axonal branching in the
 periphery and dendritic arborization as demonstrated by, e.g.,
 intracellular staining techniques. Furthermore, administration of NGF
 leads to increases in the synthesis of transmitter enzymes and increases
 in the synthesis of peptides in dorsal root ganglion cells. NGF also
 exerts effects on preganglionic neurons innervating sympathetic ganglion
 cells, presumably an indirect effect of its influence on the ganglion
 cells. Importantly, NGF can prevent death of responsive neurons pursuant
 to mechanical, chemical and immunological insults. When NGF deprivation is
 induced by axotomy or administration of antisera, atrophy and reduction in
 the synthesis of transmitter enzymes occur. Furthermore, when autoimmunity
 to NGF is induced in rats, guinea pigs and rabbits, there is massive death
 of sympathetic ganglion cells over a period of several months in animals
 that generate high antibody titers. Finally, even in adulthood, several
 neuronal populations in the peripheral and central nervous system respond
 to transection of their axons by atrophy, reductions in transmitter
 synthesis and significant degrees of cell death. Taken together, all of
 these finding in vivo suggest that trophic factors act chronically in the
 mature animal to maintain normal function. Therefore, trophic deficiency
 is probably an important mechanism in disease states of adulthood [see
 Snider, W. D. and Johnson, Jr., E. M., "Neurotrophic Molecules," Annals of
 Neurology 26:489-506 (1989) and references cited therein].
 Other neurotrophic molecules characterized thus far influence various other
 neuronal populations. The existence of different patterns of specificity
 suggests that there may be a multitude of neurotrophins with different
 specificities and activities. As the molecules occur in minimal
 quantities, their isolation is a cumbersome and time-consuming effort.
 The discovery of neurotrophic factors has obvious implications with respect
 to neurodegenerative diseases. Indeed, it has been hypothesized that the
 lack of neurotrophic factors is responsible for the degeneration of
 selective neuronal populations as it occurs in Parkinson's disease,
 Alzheimer's disease and amyotrophic lateral sclerosis, and that
 application of corresponding neurotrophic factor might prevent neuronal
 degeneration [Appel, S. H., "A unifying hypothesis for the cause of
 amyotrophic lateral sclerosis, parkinsonism, and Alzheimer's disease,"
 Ann. Neurol. 10:499-505 (1981)]. In particular, as NGF is a trophic factor
 for the population of basal forebrain cholinergic neurons which
 degenerates in Alzheimer's disease, it has been speculated that NGF may be
 useful in the treatment of this disease.
 Classical neuropharmacology attempts to influence mechanisms related to
 neuronal impulse flow and transmission at the synapse. Currently-used
 drugs and available pharmacological tools do not affect the structural
 features of the central nervous system. Moreover, there is a lack of
 compounds that are able to promote regeneration, plasticity and
 maintenance of structural integrity of selected neuronal systems. An
 increased understanding of the properties of neurotrophic factors is
 virtually certain to lead to the development of a new,
 structurally-oriented neuropharmacology. In particular, neurotrophic
 factors shall undoubtedly prove useful in the treatment of
 neurodegenerative diseases associated with structural disintegration of
 selected neuronal systems of brain areas.
 One of the more exciting features of neurotrophic molecules from a clinical
 standpoint is their ability to promote cell survival after a variety of
 insults. For example, it has been shown that NGF has the ability to save
 neurons that would ordinarily die after mechanical injury. These injuries
 have been most commonly produced by transecting the axons of sympathetic
 and dorsal root ganglion cells or basal cholinergic forebrain neurons.
 Such injuries separate the soma from contact with targets and presumably
 cause neurons to degenerate because of loss of trophic support, although
 other mechanisms may be involved. In every circumstance in which axons of
 a responsive neuronal population have been transected, NGF has saved at
 least some neurons from degenerating. NGF works after systemic
 administration for peripheral neurons, as well as after local application
 to axon tips, and is effective after intraventricular administration for
 neurons within the central nervous system. Another neurotrophic molecule,
 FGF, is also effective in some of these same paradigms. This ability to
 prevent cell death after injury is obviously relevant to the problem of
 promoting neural regeneration [see Snider and Johnson, supra, at 498 et
 seq.].
 Neurotrophins have also been shown to save neurons after exposure to
 certain toxins. For example, the concomitant administration of NGF with
 6-hydroxydopamine (which is presumed to act by destroying sympathetic
 nerve terminals, thereby interfering with the uptake of NGF from a target)
 can completely prevent the death of cells that occurs upon administration
 in early postnatal life. In addition, NGF has been shown to save neurons
 after administration of vinblastine and coichicine, which inhibit
 axoplasmic transport. Further, NGF can partially prevent the cell death in
 dorsal root ganglia caused by administration of the sensory toxin
 capsaicin to newborn animals.
 Neurotrophins are also implicated in a number of different ways with an
 organism's maintenance of healthy neuronal function. For example, NGF has
 been shown to suppress primary infection of dorsal root and sympathetic
 ganglion cells by herpes simplex type I virus; NGF is believed to suppress
 the expression of gene products necessary for viral replication. In
 addition, lymphocytic infiltration and destruction of sympathetic ganglia
 induced by administration of guanethidine and its analogues (resulting in
 autoimmune attack) is completely prevented by concomitant administration
 of NGF. The presumed mode of action is by suppressing the expression of
 the antigen on the ganglion cell surface, leading to the suggestion that
 another physiological role of trophic factors in adult animals may be to
 maintain immunological silence of irreplaceable neurons [Snider et al.,
 supra, at 499].
 Unfortunately, several formidable obstacles remain to be overcome before
 neurotrophic peptides can be of widespread clinical utility. First,
 sufficient quantities of the neurotrophins must be available; recombinant
 DNA technology will be required to engineer expression vectors that
 produce large quantities of biologically active factors. Further, the
 practical difficulties of and limitations on the administration and
 delivery of such molecules must be overcome. In order to be able to reach
 neuronal populations in the brain, neurotrophic factors would have to be
 given intracerebrally, as these proteins do not cross the blood-brain
 barrier. In human patients in particular, there would only be limited
 options for administration of neurotrophins per se. Neurotrophic factors
 purified from natural sources or produced by recombinant techniques could
 potentially be chronically infused into the brain with the help of
 mechanical pump devices; however, subcutaneous pumps are relatively
 complex devices necessitating surgical intervention, and stability of the
 active proteins during storage in these pump devices would be expected to
 necessitate special preparations. It is encouraging that local
 administration of NGF to the distal parts of injured neurons enhances
 survival and regeneration, thus suggesting the potential in some
 situations for local administration; nonetheless, even in those instances,
 the use of some sort of prosthetic device appears necessary. An
 alternative method of administration would involve the use of slow-release
 intracerebral implants containing the active protein embedded in a
 biodegradable polymer matrix. At this time, existing polymers provide
 stable release rates of only several weeks.
 While administration of modified neurotrophin molecules or active fragments
 thereof may ultimately provide a solution to the problem of providing
 therapeutic agents with neurotrophic activities, at this time very little
 is known about the possibility of producing active fragments of
 neurotrophic factors. Accordingly, it has been suggested that perhaps the
 best long-range hope for this class of agents lies in understanding in
 detail their interaction with their receptors and the molecular mechanisms
 of their trophic and survival-promoting actions; this may allow the design
 and use of low-molecular-weight drugs that mimic the effects of trophic
 factors and that can be administered in more traditional and practical
 ways [Snider, supra, at 499]. The identification of agents that modify
 components of neurotrophin activity is in and of itself a valuable
 contribution to the art, in view of the substantial present utility of
 such agents in obtaining a clearer understanding of the molecular
 mechanisms involved and in the design of novel therapeutic agents.
 At least two types of proteins are apparently involved in the formation of
 functional receptors for neurotrophin growth factors. These are the low
 affinity NGF receptor protein (p75-NGFR) [Chao, M. V. et al., "Gene
 transfer and molecular cloning of the human NGF receptor", Science
 232:518-521 (1986); Radeke, M. J., et al., "Gene transfer and molecular
 cloning of the rat nerve growth factor receptor: a new class of
 receptors"; Nature 325:593-597 (1987)] and products of trk-related
 proto-oncogenes [Hempstead, B. L. et al., "High-affinity NGF binding
 requires coexpression of the trk proto-oncogene and the low-affinity NGF
 receptor," Nature 344:339-341 (1990)]. The trk gene products, but not the
 p75-NGFR, exhibit protein kinase activity. Individual trk receptors bind
 to and stimulate tyrosine phosphorylation of different subsets of
 neurotrophins. Trk binds to NGF but not BDNF, trkB binds BDNF but not NGF.
 NT-3 is capable of interacting with trk and trkB receptors and with trkC.
 The interaction of NT-3 with multiple trk receptors may allow this factor
 to control the survival of populations of neurons expressing different trk
 gene products.
 U.S. Pat. No. 4,555,402 to Matsuda et al., the entire disclosure of which
 is hereby incorporated by reference, discloses the isolation of a
 physiologically-active substance denominated as K-252. This compound is
 described as having antiallergic and antihistamine-releasing activities.
 U.S. Pat. No. 4,923,986 to Murakata et al., the entire disclosure of which
 is also hereby incorporated by reference, discloses a class of derivatives
 of K-252 represented by the general formula
 ##STR1##
 wherein W.sub.1, W.sub.2, R.sup.1, R.sup.2, R.sup.3, R.sup.4, X and Y
 represent various substituents. The compounds are physiologically active
 substances that inhibit protein kinase C and exhibit an antitumor
 activity.
 U.S. Pat. No. 4,877,776 to Murakata et al., the entire disclosure of which
 is also hereby incorporated by reference, discloses another class of
 derivatives of K-252 represented by the general formula
 ##STR2##
 wherein R.sup.1 and R.sup.2 are independently H or OH, X represents COOH,
 COOR or CH.sub.2 OH; Y represents H, R or COR; and Z represents OH, OR or
 SR, in which R represents lower alky. These derivatives are described as
 exhibiting C-kinase inhibitory activity, and were expected to be useful as
 an active ingredient of antitumor agents, etc.
 Of the compounds disclosed in the aforementioned U.S. patents, two have
 been known for the longest time and have in particular become the subjects
 of substantial research scrutiny. K-252a and K-252b, two related
 alkaloid-like compounds from microbial origin known to interfere with
 protein kinase activities in cell-free systems, have been found to inhibit
 several biological actions of NGF [Nakanishi, S. et al., "K-252a, a novel
 microbial product, inhibits smooth muscle myosin light chain kinase," J.
 Biol. Chem. 263:6215-6219 (1986); Kase, H. et al., "K-252 compounds, novel
 and potent inhibitors of protein kinase C and cyclic nucleotide-dependent
 protein kinases," Biochem. Biophys. Res. Commun. 142:436440 (1987);
 Koizumi, S. et al., "K-252a: a specific inhibitor of the action of nerve
 growth factor on PC12 cells," J. Neurosci. 8:715-721 (1988); Matsuda, Y.
 and Fukuda, J., "Inhibition by K-252a, a new inhibitor of protein kinase,
 of nerve growth factor-induced neurite outgrowth of chick embryo dorsal
 root ganglion cells," Neurosci. Lett. 87:295-301 (1989)]. K-252a prevents
 the NGF induced morphological transformation of proliferating PC12
 pheochromocytoma cells into neuron-like cells and inhibits the NGF
 stimulated, but not the basic fibroblast growth factor (bFGF) or epidermal
 growth factor (EGF) stimulated phosphorylation of selected proteins
 [Hashimoto, S., "K-252a, a potent protein kinase inhibitor, blocks nerve
 growth factor-induced neurite outgrowth and changes in the phosphorylation
 of proteins in PC12h cells," J. Cell. Biol. 107:1531-1539 (1988); Sano, M.
 et al., "A nerve growth factor-dependent protein kinase that
 phosphorylates microtubule-associated proteins in vitro: possible
 involvement of its activity in the outgrowth of neurites from PC12 cells,"
 J. Neurochem. 55:427-435 (1990)]. Thus, to date compounds as described in
 the aforementioned U.S. patents have been shown to have only an inhibitory
 affect on neurotrophin activity.
 The development of non-peptide agonistic molecules for neurotrophic factors
 which pass the blood-brain barrier, while acknowledged as theoretically
 possible, has heretofore been considered potentially to prove a herculean
 task [Hefti, F. et al. (1989), supra, at 525]. It would therefore be
 highly desirable to identify low-molecular-weight agents which modulate
 (and, most desirably, potentiate) neurotrophin activity, both for purposes
 of elucidating the molecular mechanisms of neurotrophin action and as
 useful therapeutic agents for treatment of, e.g., neurodegenerative
 diseases.
 It is an object of the present invention to provide compositions for use in
 potentiation of neurotrophin activity, as well as methods for the
 preparation and use thereof.
 SUMMARY OF THE INVENTION
 In accordance with the present invention, there are provided compositions
 and methods for use in the potentiation of neurotrophin activity. Pursuant
 to one aspect of the present invention, the compositions comprise an
 effective amount of at least one K-252 compound, as hereinafter defined.
 One preferred class of such compounds is represented by general formula I
 ##STR3##
 in which W.sub.1, W.sub.2, R.sup.1, R.sup.2, R.sup.3, R.sup.4, X and Y are
 as hereinafter defined. In particular, selective potentiation of the
 activity of neurotrophin-3 (NT-3) has been demonstrated using particularly
 preferred compounds of general formula I in which W.sub.1, W.sub.2,
 R.sup.1, R.sup.2, R.sup.3 and R.sup.4 are all hydrogen, Y is OH and X is
 -COOCH.sub.3 (K-252a) or --COOH (K-252b). In accordance with another
 aspect of the invention, neurotrophin activity is modulated by
 administration of an effective amount of at least one compound which
 potentiates neurotrophin activity.

DETAILED DESCRIPTION OF THE INVENTION
 The role of neurotrophic factors in development and adult function of
 neurons of the mammalian brain, in particular of cholinergic and
 dopaminergic neurons which degenerate in human neurodegenerative diseases,
 has been the subject of considerable research. In the course of research,
 it was first determined that K-252a and K-252b inhibit NGF mediated
 actions on cholinergic neurons in cell culture. Both compounds at
 relatively high dosages were found to completely and selectively prevent
 the trophic action of NGF on these cells, as reflected by an increase in
 the activity of the cholinergic marker enzyme choline acetyltransferase
 (ChAT).
 In addition to the inhibitory effects observed at higher dosages, it has
 now surprisingly been determined that compounds of general formula I, such
 as K-252a and K-252b, also potentiate the activity of neurotrophins in a
 heretofore unobserved manner. This discovery makes possible not only a
 more comprehensive understanding of the molecular mechanisms of
 neurotrophin action, but also a therapeutic potentiation of neurotrophin
 action using compositions which may be administered via conventional
 routes for low-molecular-weight therapeutic agents. In addition, the
 recognition that neurotrophin activity may be potentiated by
 administration of a non-peptide active agent enables the development of a
 hitherto unanticipated realm of neuropharmacology.
 Pursuant to one aspect of the present invention, there is provided a
 composition for potentiating neurotrophin action comprising an effective
 amount of at least one K-252 compound. By K-252 compound is meant both the
 heretofore identified metabolites K-252a and K-252b, and the derivatives
 thereof described in, e.g., U.S. Pat. Nos. 4,923,986 and 4,877,776.
 A preferred class of K-252 compounds is represented by general formula I
 ##STR4##
 wherein:
 R.sup.1 and R.sup.3 are independently selected from the group consisting of
 hydrogen, lower alkyl, hydroxy, lower alkoxy, halogen and --NR.sup.5
 R.sup.6 in which each of R.sup.5 or R.sup.6 is independently hydrogen,
 lower alkyl, carbamoyl or lower alkylaminocarbonyl;
 R.sup.2 is hydrogen or amino;
 R.sup.4 is hydrogen, halogen, carbamoyl, lower alkyl, amino or --CH.sub.2
 CH.sub.2 R.sup.7, in which R.sup.7 is halogen, amino, di-lower alkylamino,
 hydroxy or hydroxy-substituted lower alkylamino;
 one of W.sup.1 and W.sub.2 is hydrogen and the other is selected from the
 group consisting of hydrogen, hydroxy, lower alkoxy and lower alkylthio,
 or both W.sup.1 and W.sub.2 are combined together to represent oxygen;
 X is hydrogen, --COOH, lower alkoxycarbonyl,
 ##STR5##
 in which R.sup.8 and R.sup.9 are independently hydrogen, lower alkyl or
 hydroxysubstituted lower alkyl, or R.sup.8 is hydrogen and R.sup.9 is
 hydroxy, --CH.sub.2 A in which A is hydroxy, azido, lower alkylthio, lower
 alkylsulfenyl, or:
 ##STR6##
 wherein R.sup.10 and R.sup.11 are independently selected from the group
 consisting of hydrogen, lower alkyl, allyl, carboxylic acid-substituted
 lower alkyl, dihydroxy-substituted lower alkyl, a residue of an
 .alpha.-amino acid in which the hydroxy of the carboxylic acid is removed
 and lower alkoxycarbonylsubstituted lower alkyl, or R.sup.10 and R.sup.11
 are combined together to form --CH.sub.2 CH.sub.2 --B-CH.sub.2 CH.sub.2 --
 in which B is --CH.sub.2 --, --NH--, --S-- or --O, --N.dbd.CH--NR.sub.2
 (wherein R is lower alkyl), --O--COCH.sub.2 CH.sub.2 CO.sub.2 H,
 ##STR7##
 or --C.dbd.N-R.sup.12 in which R.sup.12 is hydroxy, amino, guanidino or
 2-imidazolylamino; and
 Y is hydroxy, lower alkoxy, or carbamoyloxy; or X and Y are combined
 together to form, as --X--Y--, O=, --CH.sub.2 O, --CH.sub.2 OCOO,
 --CH.sub.2 O--CS--O--, --CH.sub.2 --NR.sup.13 --CO--O-- in which R.sup.13
 is hydrogen, lower alkyl, allyl, formylmethyl, --CH.sub.2 CH(OH)--CH.sub.2
 OH or --CH.sub.2 CH.dbd.N--NHC(NH.sub.2).dbd.NH, --CH.sub.2 --NH--CS--O--,
 --CH.sub.2 --O--SO--O-- or:
 ##STR8##
 wherein R.sup.14 is lower alkyl or lower alkylthio.
 This preferred class of compounds embraces K-252 compounds as described in,
 e.g., the aforementioned U.S. Pat. Nos. 4,923,986 and 4,877,776, as well
 as the naturally-occurring K-252 compounds.
 In the foregoing definitions, lower alkyl includes both straight-chain and
 branched alkyl having 1 to 5 carbon atoms, and halogen includes bromine,
 chlorine, fluorine and iodine. As would be readily apparent to those
 skilled in the art, general formula I embraces K-252a, K-252b and
 derivatives thereof as described in the aforementioned U.S. Pat. Nos.
 4,877,776 and 4,923,986. Detailed methods for the preparation of the
 compounds of general formula I are provided in U.S. Pat. Nos. 4,877,776
 and 4,923,986, and a large number of these compounds are concretely
 exemplified therein. Through routine implementation of the synthetic
 methods disclosed in U.S. Pat. Nos. 4,923,986 and 4,877,776 and other
 standard techniques, a wide variety of K-252 compounds may be prepared for
 evaluation of specificity in neurotrophin potentiation activity.
 Particularly preferred compounds of general formula I for use in
 particular in potentiating the action of NT-3 are those compounds in which
 W.sub.1, W.sub.2, R.sup.1, R.sup.2, R.sup.3 and R.sup.4 are all hydrogen,
 Y is OH and X is either --COOCH.sub.3 (K-152a) or --COOH (K-252b).
 K-252b was used for most further detailed investigations, as it was
 effective over a wide range of concentrations and lacked cytotoxicity.
 K-252b has now been demonstrated to exhibit a biphasic activity profile:
 It is both a selective inhibitor of growth factors of the neurotrophin
 family and, at lower concentrations, potentiates NT-3 actions. K-252b
 enhances the trophic activity of NT-3 on primary neurons and PC12 cells
 and also stimulates the NT-3 mediated tyrosine phosphorylation of trk and
 the trk substrate phospholipase C-gammal. The stimulatory actions of
 K-252b are believed due to direct or indirect effects of this compound on
 trk signal transduction pathways.
 Compounds of general formula I (as exemplified by K-252b) selectively
 modify actions of the neurotrophin growth factor family. At nM
 concentrations, K-252b selectively potentiates the actions of NT-3; at
 .mu.M concentrations, the compound inhibits the actions of all
 neurotrophins, without interfering with transduction mechanisms of
 non-neurotrophin growth factors. Furthermore, the selective inhibitory and
 stimulatory actions of K-252b on neurotrophins are likely due to a direct
 interaction with tyrosine protein kinase activity of trk-type receptor
 proteins.
 A possible explanation for the observation of increased NT-3 effects is
 that K-252b modifies trk in a manner that this receptor interacts with
 NT-3, but not NGF, more efficiently. While K-252a does not interfere with
 binding of .sup.125 I-NGF to PC12 cells, it remains possible that the
 related K-252b might affect binding of selected neurotrophins to specific
 active sites. Such an effect could involve other proteins believed to be
 part of neurotrophin receptors, like the p75--NGFR low affinity NGF
 receptor protein.
 K-252b, besides inhibiting trk protein kinase activity, interferes with
 protein kinase C, as well as cAMP- and cGMP-dependent protein kinases with
 K.sub.i values in the 10-100 nM range as shown by in vitro assay systems.
 Given this rather broad spectrum of inhibitory actions, the selective
 inhibition of neurotrophin effects is surprising. It seems possible that
 in intact cells K-252b interacts with extracellular or transmembranal
 domains of trk proteins, without access to intracellular protein kinases.
 This possibility is supported by recent findings showing that K-252b
 inhibits the protein kinase activity of the platelet-derived growth factor
 in cell-free preparations but not in intact cells.
 While it is contemplated in accordance with the present invention that
 neurotrophin potentiating agents may be administered in conjunction with
 native and/or recombinant neurotrophins themselves in a manner as
 previously described herein, the discovery of neurotrophin potentiating
 agents (such as the low-molecular-weight compounds of general formula I)
 provide the significant advantage relative to the neurotrophic peptides
 themselves that they may be administered alone by the wide variety of
 routes heretofore employed for administration of non-peptide active agents
 in order to potentiate the activity of the patient's own neurotrophic
 factors. For example, oral, intravenous, subcutaneous, intramuscular,
 mucosal and transdermal routes of administration all would be suitable for
 use in accordance with the present invention.
 In view of the biphasic activity profile of the compounds of general
 formula I, it is important that the compounds be provided in a amount
 effective to achieve the desired neurotrophin potentiation but less than
 the amount which results in the inhibitory activity. An effective dose in
 vitro for these purposes is in the range of about 0.1-10 nM (approximately
 44.1-4,410 ng/l). Therefore, assuming uniform distribution in the human
 body and an average human body size of 70 kg (i.e., 70 liters), a dose
 projection of approximately 3-300 .mu.g is obtained. Of course, based upon
 the foregoing information, it would be well within the skill of those
 working in the pharmaceutical field to determine the optimum dosage for
 any given compound of general formula I in any particular context.
 In addition to the traditional routes of administration which have been
 practiced for centuries in the medical arts (e.g., oral and intravenous
 administration), more recently-developed techniques for administration of
 the compounds of general formula I may be employed. For transdermal
 delivery of the active agents, suitable pads or bandages are well known in
 the art. Typically, these pads comprise a backing member defining one
 exterior surface, a surface of pressure-sensitive adhesive defining a
 second exterior surface, and disposed therebetween a reservoir containing
 the active agents confined therein. Suitable transdermal delivery systems
 are disclosed in U.S. Pat. Nos. 3,731,683 and 3,797,494 to Zaffaroni and
 U.S. Pat. No. 4,336,243 to Sanvordeker et al., the entire disclosures of
 which are hereby incorporated by reference.
 Other suitable formulations would also be readily apparent to those of
 skill in the art. For example, administration may be effected
 subcutaneously or intramuscularly with slowly-dissolving pellets of
 crystalline or microcrystalline materials, or directly as a crystalline or
 microcrystalline aqueous suspension. In addition to the compounds of
 general formula I, pharmacologically acceptable salts thereof are also
 contemplated for use in accordance with the present invention. As
 indicated in U.S. Pat. No. 4,923,986, in cases where the compound of
 general formula I is an acidic compound, base addition salts can be
 formed; where the compound of general formula I is basic, acid addition
 salts can be formed. Suitable base and acid addition salts for use in
 pharmaceutical preparations are well known to those skilled in the art,
 and illustrative examples thereof are described in the aforementioned U.S.
 Pat. No. 4,923,986.
 Compounds of general formula I, such as K-252b, are also unique tools to
 study the mechanisms of action of neurotrophins and to demonstrate
 biological actions of these proteins in vivo and in vitro. Compared to the
 related compounds K-252a and staurosporine, which show complex patterns of
 activity and are cytotoxic, K-252b is a non-toxic and highly selective
 modifier of neurotrophin actions in vitro.
 The following examples will serve to illustrate the invention without in
 any way being limiting thereon.
 EXAMPLES
 The preparations of human recombinant neurotrophins were produced in a
 Chinese hamster ovary cell line according to a published procedure
 [Knusel, B. et al., "Trophic actions of recombinant human nerve growth
 factor on cultured rat embryonic CNS cells," Experimental Neurology
 110:274-383 (1990)]. The neurotrophins were purified to &gt;95% purity
 using chromatographic procedures. The neurotrophins were initially
 contained at a concentration of 0.2-1 mg/ml at pH 3 and were further
 diluted in culture medium immediately before use. Biological activity for
 all tested neurotrophin preparations was determined with chick dorsal root
 ganglion and nodose ganglion assays. Recombinant human
 des(1-3)-insulin-like growth factor-1 (des-IGF-1) was obtained from
 Genentech, South San Francisco, Calif. and initially contained in 100 mM
 sodium acetate.
 K-252b was obtained from Kyowa Hakko Kogyo Co., Tokyo, Japan. K-252a,
 KT5720, KT5823, KT5296 and staurosporine were obtained from Kamiya
 Biomedical Company (Thousand Oaks, Calif.). These compounds were dissolved
 in dimethyl sulfoxide (DMSO) at 2 mM concentrations. Aliquots of this
 solution were kept at -70.degree. C. Maximal concentration of DMSO in the
 medium was 0.01%, which was found not to affect the cultures.
 Example 1
 Inhibitory activity of K-252b
 Recombinant human NGF (rhNGF), BDNF (rhBDNF), and NT-3 (rhNT-3) and K-252b
 were added to primary cultures of fetal rat brain neurons containing
 either forebrain cholinergic or midbrain dopaminergic neurons. A broad
 range of concentrations of K-252b were tested in presence of 50 ng/ml
 rhNGF, 200 ng/ml rhBDNF and 1 .mu.g/ml rhNT-3, growth factor
 concentrations producing maximal trophic actions on cholinergic neurons.
 The trophic action was monitored by measuring the activity of ChAT, a
 parameter reflecting both survival and transmitter-specific
 differentiation of cholinergic cells.
 Primary cultures of fetal rat brain septal and mesencephalic cells were
 prepared in a heretofore known manner [Knusel et al., supra]. Briefly,
 defined areas were dissected from fetal rat brains (Wistar, E15-16,
 Charles River, Mass.). The septal area contained the cholinergic neurons
 from septum, diagonal band of Broca and nucleus basalis. The ventral
 mesencephalon containing the dopaminergic neurons of the substantia nigra
 and the ventral tegmental area was dissected and dissociated mechanically.
 The cells were plated in 16 mm multiwell plates precoated with
 polyethyleneimine (1 mg/ml, 37.degree. C., overnight), containing 0.5 ml
 modified L-15 medium supplemented with 5% heat inactivated horse serum and
 0.5% heat inactivated fetal calf serum. Modified L-15 was prepared [Knusel
 et al., "Selective and Nonselective Stimulation of Central Cholinergic and
 dopaminergic Development in vitro by Nerve Growth Factor, Basic Fibroblast
 Growth Factor, Epidermal Growth Factor, Insulin and the Insulin-like
 Growth Factors I and II," J. Neurosci. 10:558-570 (1990)] by adding
 various amino acids, vitamins, antibiotics, glucose and NaHCO.sub.3 to
 Leibovitz's L-15 medium (Gibco, Grand Island, N.Y.). Plating densities
 were 4.times.10.sup.5 cells/cm.sup.2 for basal forebrain cultures and
 3.times.10.sup.5 cells/cm.sup.2 for mesencephalic cultures. Of these
 cells, 0.5 to 1% were cholinergic or dopaminergic, respectively. Growth
 factors were typically added on the second day of culture and the cells
 were grown for 5 or more days. For ChAT assays tissue was homogenized in
 250 .mu.l of 50 mM Tris-HCl buffer, pH 6.0 with 0.3% Triton X-100.
 [1-.sup.14 C]acetylcoenzyme A (NEN) concentration was 20 .mu.M and
 specific activity 4.09 Ci/mol. To measure dopamine uptake, cultures were
 preincubated for 5 min at 37.degree. C. with 250 .mu.l incubation solution
 (5 mM glucose, 1 mM ascorbic acid in PBS) containing 1 mM pargyline.
 [.sup.3 H]dopamine (37 Ci/mmol) was then added to give a final
 concentration of 50 nM and the cultures were incubated for another 15
 minutes. Blanks were obtained by incubating cells at 0.degree. C.
 FIG. 1 illustrates levels of ChAT activity in cultures of rat basal
 forebrain treated with rhNGF, rhBDNF, or rhNT- and K-252b. Growth factors
 were added on the second day of culture and the cells were grown for 5
 days. Growth factor concentrations producing maximal elevations of ChAT
 activity were used. In FIG. 1, .largecircle. represents the control;
 .quadrature., 50 ng/ml rhNGF; .DELTA., 200 ng/ml rhBDNF; and
 .tangle-soliddn., 1 .mu.g/ml rhNT-3. The ChAT stimulation mediated by all
 three neurotrophins was inhibited by K-252b at concentrations above 200
 nM. At concentrations between 100 pM and 30 nM, K-252b potentiated the
 actions of NT-3 without affecting those of NGF or BDNF. N=4 per data
 point. Error bars represent SEMs and were omitted where they would have
 appeared smaller than the symbol.
 K-252b, at concentrations higher than 200 nM, inhibited the ChAT activity
 increase mediated by all three neurotrophins (FIG. 1). The concentration
 requirements for the inhibitions of rhNGF, rhBDNF and rhNT-3 actions
 appeared identical. The minimal concentration of K-252b which was required
 to completely abolish the responses was approximately 2 .mu.M (FIG. 1).
 The increases in CHAT activity mediated by basic fibroblast growth factor
 (bFGF), insulin, and insulin-like growth factor-1 were not affected by
 K-252b.
 BDNF, but not NGF or NT-3, trophically acts on dopaminergic neurons as
 reflected by an increase in the activity of dopamine uptake by these
 cells. Similar to its actions on cholinergic neurons, K-252b prevented the
 increase in dopamine uptake mediated by BDNF, as shown in Table 1.
 Dopamine uptake is also stimulated by other growth factors, including
 bFGF, epidermal growth factor, insulin, insulin-like growth factors-1 and
 -2. K-252b does not inhibit the stimulatory action of bFGF and insulin. As
 a additional control, des-IGF-1 was used; K-252b was found not to inhibit
 its stimulatory action on dopamine uptake (Table 1). The findings obtained
 on central cholinergic and dopaminergic neurons showed that K-252b, at
 concentrations above 200 nM, completely and selectively blocks the actions
 of all neurotrophins stimulating these cells in primary cultures, whereas
 comparable effects of non-neurotrophin growth factors are not inhibited.
 As shown in Table 1, K-252b inhibits the stimulatory action of rhBDNF, but
 not des-IGF-1 on dopamine uptake in cultures of ventral mesencephalon. In
 Table 1, dopamine uptake is given as fmol/min/culture dish. Cultures were
 grown in 24-well plates for 7 days in L-15 medium with 5% horse and 0.5%
 fetal bovine serum and treated with growth factors and K-252b from the
 second day of culture. rhBDNF, 200 ng/ml; des-IGF-1, 1 .mu.g/ml; n=4;
 *different from respective control group, p &lt;0.01 (Student's t-test).
 The apparent difference between control cultures grown in presence and
 absence of K-252b was statistically not significant. The experiment shown
 is a representative case of a total of 5 independent experiments.
 TABLE 1
 With K-252b
 Growth No K-252b (5 .mu.M)
 Factor % of % of
 Treatment Mean .+-. SEM Control Mean .+-. SEM Control
 Control 15.3 .+-. 3.6 11.4 .+-. 0.2
 rhBDNF 28.0 .+-. 0.3* 183 13.6 .+-. 0.7 119
 des-IGF-1 26.3 .+-. 1.1* 172 22.9 .+-. 1.0* 201
 Example 2
 Potentiation of NT-3 actions by K-252b on brain neurons
 Surprisingly, the detailed dose-response analysis of K-252b actions on
 cholinergic neurons revealed that the compound strongly enhanced the
 trophic effect of NT-3 on these cells at concentrations lower than those
 producing inhibitory effects. In the presence of 10-100 nM of K-252b, NT-3
 (which by itself elevated ChAT activity only by approximately 20% of the
 NGF-induced elevation) produced the same stimulatory effect as NGF (FIG.
 1). The stimulatory effects of NGF or BDNF on cholinergic neurons were not
 potentiated by these low concentrations of K-252b (FIG. 1), and similarly
 the low concentrations did not potentiate the action of BDNF on
 dopaminergic neurons.
 The detailed dose-response analysis revealed that K-252b, at 50 nM,
 increased both potency and maximal efficacy of the trophic action of NT-3
 (FIG. 2). 50 nM K-252b increased potency and efficacy of NT-3 stimulation
 of ChAT activity in cultures of rat basal forebrain. Half-maximal
 concentrations were calculated by non-linear fit of a sigmoid curve. The
 broken line represents NT-3 alone, ED.sub.50 =175.+-.72.6 ng/ml
 (mean.+-.S.E.); the solid line represents NT-3 +K-252b, ED.sub.50
 =14.3.+-.1.6 ng/ml; n=8 per symbol. Culture conditions were as described
 in connection with the results illustrated in FIG. 1.
 Example 3
 Potentiation of neurite NT-3 mediated survival of chick sensory neurons and
 neurite outgrowth of PC12 cells
 To establish generally that K-252b (at concentrations lower than those
 inhibiting neurotrophin responses) acts as a selective enhancer of NT-3,
 cultures of different neurotrophin responsive cell populations were
 tested. Survival and neurite outgrowth of chick dorsal root ganglia
 neurons (DRG) is supported by NGF, BDNF and NT-3, and the extent of the
 effect and the subpopulation supported by each factor is a function of the
 embryonic age of the animals used to prepare the cultures. In DRG cultures
 of embryonic day 9, NGF is most effective in promoting neuronal survival,
 whereas NT-3 only produces a moderate effect.
 DRGs of embryonic day 9 chicks were dissected and dissociated using
 enzymatic and mechanical procedures as described in the literature
 [Rosenthal, A. et al., "Primary Structure and Biological Activity of a
 Novel Human Neurotrophic Factor," Neuron 4:767-773 (1990)]. 1800 neurons
 were plated per well in 96-well tissue culture plates pretreated with
 polyornithine (500 .mu.g/ml) and laminin (10 .mu.g/ml). Cells were
 incubated for 48 hrs with or without growth factors and the indicated
 concentrations of K-252b. Phase-bright cells with elaborated neurites
 5.times. the diameter of the cell bodies were then counted.
 Similar to the findings obtained in primary cultures of brain cholinergic
 neurons, low concentration of K-252b (50 nM) potentiated the survival
 promoting action of a maximally effective concentration of NT-3 (FIG. 3).
 Survival of dissociated chick DRG neurons in presence of rhNGF (100 ng/ml)
 or rhNT-3 (100 ng/ml) and K-252b is illustrated in FIG. 3; concentrations
 on the horizontal axis are for K-252b. The columns represent means +SEMs.
 The number of neurons supported by the combination of NT-3 and 50 nM
 K-252b was identical to that supported by NGF alone. The same
 concentration of K-252b failed to influence survival mediated by NGF (FIG.
 3) or BDNF. High concentrations of K-252b (10 .mu.M) inhibited the action
 of NGF on sensory neurons, as found for cholinergic neurons (FIG. 3).
 Similar results were also seen in cultures of dissociated chick
 sympathetic neurons.
 PC12 cells were grown as described earlier [Kaplan et al., 1990, 1991a, b].
 To assess effects on neurite outgrowth, cells were incubated for 48 hours
 with K-252b (50 nM) and NT-3 or NGF (50 ng/ml). Neurites were scored if
 they were a length of one cell body or more.
 To measure the status of trk tyrosine phosphorylation, PC12 cells were
 lysed and immunoprecipitated with anti-trk serum. The trk proteins were
 subjected to 7.5% SDS-PAGE and analyzed by immunoblotting with
 anti-phosphotyrosine antibodies as described in detail in the literature
 [Kaplan, D. R. et al., "PDGF beta receptor stimulates tyrosine
 phosphorylation of GAP and association of GAP with a signaling complex,"
 Cell 61:125-133 (1990)]. Tyrosine phosphorylation of cellular proteins was
 assayed by probing Western blots of lysates of PC12 cells incubated with
 K-252b and NT-3 or NGF with anti-phosphotyrosine antibodies. Phospholipase
 C gammal was identified in these blots as described elsewhere [Vetter,
 M.L. et al., "Nerve growth factor rapidly stimulates tyrosine
 phosphorylation of phospholipase C-gammal by a kinase activity associated
 with the product of trk protooncogene," Proc. Natl Acad. Sci. USA
 88:5650-5654 (1991)].
 Experiments on PC12 cells confirmed observations made on primary neuron
 cultures. Treatment with K-252b stimulated NT-3 induced neurite outgrowth
 and trk tyrosine phosphorylation in PC12 cells while inhibiting NGF
 induced effects. PC12 cells were incubated for 48 hours with K-252b (50
 nM) and NT-3 or NGF (50 ng/ml). FIG. 4A illustrates the % cells with
 neurites after 48 hours incubation. Neurites were scored if they were a
 length of one cell body or more. PC12 cells were subsequently lysed and
 immunoprecipitated with anti-trk serum. trk proteins were subjected to
 SDS-PAGE and analyzed by immunoblotting with anti-phosphotyrosine
 antibodies (FIG. 4B). Lane 1, NT-3 +K-252b; lane 2, NT-3; lane 3, NGF
 +K-252b; lane 4, NGF. For FIG. 4C, PC12 cells were incubated for 1 hour in
 K-252b (50 nM or 10 .mu.M) and with NT-3 or NGF for 5 minutes prior to
 lysis; other methods were the same as described for FIG. 4B. Lane 1,
 NT-3+10 .mu.M K-252b; lane 2, NT-3.+-.50 nM K-252b; lane 3, NT-3; lane 4,
 NGF+10 .mu.M K-252b; lane 5, NGF+50 nM K-252b; lane 6, NGF; lane 7,
 untreated control.
 PC12 cells incubated for 48 hours in the presence of NT-3 (50 ng/ml) and
 K-252b (50 nM) showed significantly more neurite outgrowth activity than
 cells incubated with NT-3 alone (FIG. 4A). At the concentration of K-252b
 used in this experiment, NGF-induced neurite outgrowth was reduced
 approximately three-fold, whereas in the primary neuron cultures no
 significant inhibition was detected at this concentration (FIG. 1).
 Example 4
 Effects of K-252b on tyrosine phosphorylation of trk
 The fact that K-252b inhibits various protein kinases in cell free systems
 and the recent discovery that trk proto-oncogenes are involved in the
 formation of high affinity neurotrophin receptors suggested that K-252b
 exerts its neurotrophin inhibitory and stimulatory actions by directly
 interfering with protein kinases of the trk protein family. NGF has been
 shown to stimulate the tyrosine phosphorylation of pl40 trk within minutes
 of addition to PC12 cells. NT-3 induces only low levels of trk tyrosine
 phosphorylation and neurite outgrowth in these cells (FIG. 4A). PC12 cells
 which were exposed to NT-3 or NGF for 48 hours in the presence or absence
 of K-252b (FIG. 4A) were also analyzed for pl40 trk tyrosine
 phosphorylation (FIG. 4B). NGF strongly stimulated trk tyrosine
 phosphorylation whereas no phosphorylation was detectable with NT-3 alone.
 However, a clear increase in trk tyrosine phosphorylation was seen in
 cells grown in presence of NT-3 and 50 nM K-252b (FIG. 4B). Similar
 effects were observed when cells were acutely treated for 1 hour with
 K-252b followed by 5 minutes NT-3 (FIG. 4C). Stimulation of tyrosine
 phosphorylation by NT-3 alone was minimal but was greatly enhanced by the
 simultaneous presence of K-252b. NGF produced a pronounced increase of trk
 phosphorylation and K-252b at 10 .mu.M partially inhibited this effect.
 Cells treated with the inhibitor alone and without NT-3 were identical to
 untreated controls. In contrast to the pronounced effects of K-252b on trk
 tyrosine phosphorylation mediated by neurotrophins, K-252b failed to
 influence similar responses induced by epidermal growth factor and basic
 fibroblast growth factor on their corresponding receptors.
 Tyrosine phosphorylation of cellular proteins was examined in PC12 cells
 treated with NT-3 or NGF in presence of K-252b. The tyrosine
 phosphorylation of phospholipase C gamma-1, a direct target of the trk
 tyrosine kinase and several other cellular proteins was inhibited by
 K-252b in NGF treated cells. In contrast, the NT-3 mediated tyrosine
 phosphorylation of these proteins in PC12 cells was enhanced by 50 nM of
 K-252b.
 Example 5
 Human trk and trkB were expressed in Sf9 insect cells transfected using a
 baculovirus system. The trk proteins were immunoprecipitated as described
 above and the precipitates were incubated with 20 .mu.Ci [gamma-.sup.32
 P]ATP, 10 mM MnCl.sub.2, 20 mM Tris, pH 7.4, for 5 min at 25.degree. C. in
 the presence of increasing amounts of K-252b or control solution.
 Phosphorylated proteins were analyzed as described for FIG. 4B. The
 tyrosine kinase activity of the trk and trkB proteins was activated in the
 absence of ligand, a common observation for receptor tyrosine kinase
 produced in the baculovirus system. The trk and trkB proteins were 25%
 pure as assayed by SDS-PAGE.
 A direct action of K-252b on trk was demonstrated using partially purified
 recombinant trk proteins. 10 .mu.M K-252b completely prevented tyrosine
 phosphorylation of both, trk and trkB in a cell-free system (FIG. 5).
 K-252b interacts directly with the trk and trkB protein. Human trk and
 trkB were expressed in Sf9 insect cells transfected using a baculovirus
 system. trk proteins were immunoprecipitated and the precipitates were
 incubated with [gamma.sup.32 P]ATP in the presence of increasing amounts
 of K-252b or control solution.
 Example 6
 Structural Requirements for Potentiation Effect
 To assess the structural features of the compounds of general formula I
 which are responsible for selective potentiation of NT-3 and to decide
 whether these features are different from the ones mediating neurotrophin
 inhibition, K252b and various structural relatives were studied. In
 addition to K-252b ("K2b"), K-252a ("K2a"), KT5720 ("K57"), KT5823
 ("K58"), KT5296 ("K59") (within the scope of general formula I):
 ##STR9##
 (outside the scope of general formula I) were found to inhibit the action
 of central cholinergic neurons. These compounds were accordingly tested
 for possible NT-3 potentiation at lower concentrations than necessary for
 NGF inhibition. Culture conditions were as described in Example 1.
 Cultures were untreated (controls) or treated with 200 ng/ml NT-3 and
 increasing concentrations of the inhibitors. The findings are shown in
 FIG. 6, in which "Stau", "K2a", "K2b", "K57", "K58", "K59" represent,
 respectively, staurosporine, K-252a, K-252b, KT5720, KT5823, and KT5296.
 K-252a, KT5720 and KT5823 induced potentiation similar to K-252b.
 Staurosporine and KT5296 were ineffective. The absence of NT-3
 potentiating effects of staurosporine and KT5296, which also selectively
 inhibit NGF, demonstrates that the structural requirements for NGF
 inhibition and for NT-3 potentiation are different from one another.
 Moreover, this result confirms that compounds of general formula I exhibit
 an unexpected activity relative to compounds of remarkably similar
 structure but lacking the characteristic five-membered oxygen-containing
 ring of general formula I. The studies furthermore revealed that addition
 of hydroxy-propyl substituent in the aromatic ring system results in loss
 of the potentiating property.
 While there have been shown and described the fundamental novel features of
 the invention, it will be understood that various omissions, substitutions
 and changes in the form and details of the invention illustrated may be
 made by those skilled in the art without departing from the spirit of the
 invention. It is the intention, therefore, to be limited only as indicated
 by the scope of the following claims.