WO2005094824A1

WO2005094824A1 - Methods of enhancing cancer therapy by protecting nerve cells

Info

Publication number: WO2005094824A1
Application number: PCT/US2005/009528
Authority: WO
Inventors: Qun Lu; Shiloh B. Jones; Hope Y. Lu
Original assignee: East Carolina University
Priority date: 2004-03-25
Filing date: 2005-03-24
Publication date: 2005-10-13

Abstract

The present invention provides methods of treating cancer in a subject with at least one antineoplastic compound, the improvement comprises administering to the subject an activator or inhibitor of a Rho family signaling pathway protein in an amount effective to inhibit neuronal impairment in the subject caused by the antineoplastic compound. Methods of screening for compounds useful for inhibiting neuronal impairment caused by administering an antineoplastic compound are also provided.

Description

METHODS OF ENHANCING CANCERTHERAPY BY PROTECTING NERVE CELLS

Field of the Invention The present invention concerns methods of enhancing cancer therapy by ameliorating cell damage caused by anti-cancer agents and further concerns methods of screening compounds that may ameliorate cell damage caused by anti-cancer agents.

Background of the Invention The development of functional circuitry in the central nervous system is an intricate process that relies, in part, on the morphological complexity of resident neurons. Early in development, neuronal precursors leave the ventricular zone and migrate toward their destination underneath the pial surface, where they begin to differentiate in the cortical plate. Polarity is established during the process of differentiation and the neuron sends out two types of projections, a single axon, which conveys output and a number of dendrites, which collect input from other neurons. Dendrites act as integrators of synaptic input, thus the process of dendrogenesis determines the number and pattern of synapses that are received by each mature neuron (McAllister, 2000). Development of the dendritic tree is a dynamic series of events that results in the formation of a complex and highly ordered structure through abundant remodeling and reorganization of the microfilament and microtubule cytoskeletons. These processes are regulated by a variety of extracellular signals, which eventually converge on signaling pathways that effect cytoskeletal reorganization (Luo, 2000). The proper development of axons and dendrites plays a role in synapse formation, and therefore the establishment of appropriate neuronal circuitry. If these developmental events are disrupted, e.g., by genetic alteration, metabolic disturbance, or by drug induced toxicities, the result could be a devastating dendritic impairment that leads to mental retardation in children or cognitive malfunction in adults (Marin-

Padilla, 1976; Becker et al., 1986; Comery et al., 1997 and Kaufmann et al., 2000). The Abl family of non-receptor tyrosine kinases is a link in signal transduction pathways that promote cytoskeletal rearrangement. Mammalian members Abl (also known as Abll), Arg (Abl-related gene, also known as Abl2) and Drosophila abl (D- Abl) share a conserved structure and have been suggested to function in cell adhesion and actin cytoskeletal reorganization. (In a neuronal context, Abl kinases have been shown to play a role in neuralation, as abllarg double null mice exhibit defective neural tube morphology and gross alterations in neuroepithelial actin cytoskeleton structure (Koleske et al., 1998). Abl kinases are additionally involved in modulating actin dynamics in axonogenesis, growth cone motility and have been shown to stimulate neurite outgrowth in primary neuronal cultures (Zukerberg et al., 2000; Woodring et al., 2002). Abl localization has also been demonstrated in the growth cones of primary hippocampal neurons, where it interacts with δ-catenin, a dendrite specific Abl substrate that has recently been shown to affect dendrite outgrowth and branching (Kim et al., 2002; Lu et al., 2002; Martinez et al., 2003). In addition, Abl kinases are localized to both presynaptic and postsynaptic sites in mature neurons and at the neuromuscular junction (Finn et al., 2003; Moresco et al., 2003). Abl kinase activity regulates actin dependent processes through interactions with several protein families, including Rho GTPase family members Racl and Rho A (Nan Etten, 1999; Lanier and Gertler, 2000). Abl is demonstrated to regulate RhoA and Racl through functional interactions with Trio, a guanine nucleotide exchange factor (GEF), with discrete domains for both GTPases (references). Most recently, Abl kinases have also been implicated in decreasing RhoA activation through phosphorylation of an upstream Rho regulator, pl90RhoGAP (Moresco and Koleske, 2003). Signaling pathways involving the Rho family of small GTPases have been shown to mediate distinct actin cytoskeleton reorganization events in several cell types, including neurons and have been proposed to be key mediators of dendritic development. In particular, expression of RhoA mutants has demonstrated that RhoA is involved in the negative regulation of dendrite outgrowth (references). Studies in several systems have shown that expression of constitutively active RhoA inhibits dendrite growth and induces neurite retraction. Conversely, RhoA null mutations promote dendrite outgrowth and cause overextension of dendritic processes. Active RhoA can initiate cell extensions retraction by acting on Rho associated kinase/pl60/ROCK. ROCK is further demonstrated to negatively regulate neurite initiation in a RhoA dependent manner as demonstrated by studies showing that RhoA mediated process retraction can be prevented by inhibition of ROCK activity (Da Silva et al., 2003; Hirose et al., 1998; Bito et al., 2000; Nakayama et al., 2000). Summary of the Invention The present invention provides methods of enhancing anti-cancer therapy by ameliorating cell damage caused by antineoplastic compounds and methods of screening compounds that may ameliorate cell damage caused by antineoplastic compounds. In one embodiment, the present invention provides methods of treating cancer in a subject with at least one antineoplastic compound, the improvement comprises administering to the subject an activator or inhibitor of a Rho family signaling pathway protein in an amount effective to inhibit neuronal impairment in the subject caused by the antineoplastic compound. In other embodiments, the present invention provides methods of treating cancer in a subject with imatinib or an analog thereof or a pharmaceutically acceptable salt thereof, the improvement comprises administering to the subject a Rho kinase inhibitor in an amount effective to enhance the antineoplastic activity or reduce the neurotoxicity of the imatinib or analog thereof or a pharmaceutically acceptable salt thereof. In further embodiments, the present invention provides the use of an active compound as described herein for preparation of a medicament for carrying out a method as described herein. In still other embodiments, the present invention provides methods of screening for compounds useful for inhibiting neuronal impairment caused by administering an antineoplastic compound comprising contacting a neuronal cell with a test compound, in the presence of an Abl family tyrosine kinase inhibitor, wherein the contacting step is carried out in vitro; and then determining whether the test compound affects dendrogenesis influenced by Abl family tyrosine kinase inhibition; a positive effect on the dendrogenesis influenced by the Abl family tyrosine kinase inhibition indicates that the test compound is useful for inhibiting neuronal impairment caused by administering an antineoplastic compound. The foregoing and other objects and aspects of the present invention are explained in greater detail in the drawings herein and the specification set forth above. Brief Description of the Drawings Figure 1 illustrates that inhibition of Abl family tyrosine kinase activity disrupts dendrogenesis independent of axonogenesis. A: Developmental progression of cultured hippocampal neuron from 0.5 day in vitro (DIN) through 7 DIN. B: Treatment with the small molecule inhibitor STI571 from 4 DIN inhibits Abl family kinase activity, leading to a simplification of dendritic morphology, while axonal development proceeds normally. Arrows point to dendrites; arrowheads indicate axons. Scale bar = 12 mm. Figure 2 illustrates that inhibition of Abl family tyrosine kinase activity alters the developmental dynamics of dendrogenesis. Hippocampal neurons grown in culture for 4.5 days were incubated in the absence (Control) or presence (STI571) of Abl inhibitor for 48 hours. Time-lapse images were captured every 15 seconds for 15 minutes. Only 6 frames (every 3 minutes) are shown here. Arrows point to the axons while arrowheads indicate the dendrites. The axon motility is obvious in both control and STI571 treated neurons. Dendritic growth cones in control neurons show an active bifurcation and motility, but STI571 treated neurons shows a reduced bifurcation. See supplemental video images for details. Figure 3 illustrates that inhibition of Abl family tyrosine kinase activity disrupts neuronal polarity. A: Immunofluorescent images of control neurons labeled with (a) FITC phalloidin (asterisk shows growth cone); (b) anti-MAP2; (c) anti-Tau B: Hippocampal neurons 4 DIN treated with 3mM STI571 for 48 hours stained with FITC phalloidin (a) and immunostained for (b) anti-MAP2; (c) anti-Tau. Note disruption of MAP2 and Tau segregation upon Abl kinase inhibition. Arrows point to dendrites; arrowheads indicate axons. Scale bar = 12 mm. Figure 4 illustrates that Abl family tyrosine kinase activity influences dendrite formation. Quantification of primary and secondary dendrite outgrowth in neurons incubated with DMSO, STI571 or transfected with a constitutively active Abl kinase construct. *p< .0\. Figure 5 illustrates that inhibition of Abl family tyrosine kinase activity leads to an increase in GTP-bound RhoA without affecting Racl activity. Lysates of 7 DIN cortical cultures treated with DMSO as control or 3mM STI571 for 48 hours were incubated with GST-RBD glutathione beads to pull down GTP-bound RhoA as an indication of RhoA activity. Bound proteins were analyzed by western blot with an antibody against RhoA. Likewise, GST-PBD glutathione beads used to pull down GTP-bound Racl were immunoblotted with an antibody against Racl to indicated Racl activity. Figure 6 illustrates that ROCK inhibition suppresses STI571 effect on dendrogenesis. Immunofluorescence of anti-MAP2 antibody and Rhodamine phalloidin staining of untreated hippocampal (panel 1), neurons treated with STI571

(panel 2), Y-27632 to inhibit ROCK (panel 3), and STI571 + Y-27632 (panel 4).

Scale bar = 12 mm. Figure 7 illustrates that RhoA activation suppresses CA-Abl effect on dendrogenesis. Immunofluorescent images of hippocampal neurons transfected with CA-RhoA (panel 1), CA-Abl (panel 2), and CA-RhoA + CA-Abl (panel 3). Top: GFP fluorescence. Bottom anti-Abl. Scale bar = 12 mm. Figure 8 illustrates that the CA-Abl effect on dendrogenesis is dependent upon the reorganization of actin cytoskeleton but is independent of the stability of microtubules. A: Immunofluorescence of anti-Abl antibody (top panel) and Rhodamine phalloidin (bottom panel). Control cells and hippocampal neurons transfected with CA-Abl were treated with 5mM Latrunculin A for 24 hours to depolymerize actin filaments. They were also treated with lOOnM Jasplakinolide for 5 hours to stabilize actin filaments. B: Immunofluorescence of anti-Abl antibody (top panel) and anti-MAP2 (bottom panel). Control neurons or CA-Abl expressing cells were incubated with or without the presence of 5 mM Nincristine for 5 hours. Scale bar = 12 mm.

Detailed Description of Embodiments of the Present Invention The foregoing and other aspects of the present invention will now be described in more detail with respect to embodiments described herein. It should be appreciated that the invention can be embodied in different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used in the description of the invention and the claims set forth herein, the singular forms "a," "an," and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise. All publications, patent applications, patents and other references cited herein are incorporated by reference in their entireties. As used herein, "antineoplastic compound" refers to a compound capable of inhibiting the growth or spread of an abnormal growth such as cancer, tumors and the like. Antineoplastic compounds can be antimetastatic compounds. Examples of antineoplastic compounds include, but are not limited to, alkylating agents, antimetabolites, natural products and their derivatives, hormones and steroids. As used herein, "activator" refers to a compound that activates, induces, stimulates, increases or prolongs the activity of a compound or mechanism. As used herein, "inhibitor" refers to a compound that inhibits, prevents, decreases or stops the activity of a compound or mechanism. As used herein, "amount effective" refers to an amount effective to provide a benefit to the subject including the slowing of the progression of undesirable effects. As used herein, "neurons" refer to excitable cells in the central and peripheral nervous system specialized for transmission of chemical and electrical signals. As used herein, "neuronal impairment" refers to deleterious effects on neurons including, but not limited to, neuronal cell death, impaired dendrogenesis, actin- cytoskeletal reorganization and chemotherapy-induced DNA damage. As used herein, "toxic" refers to the capability of eliciting a deleterious effect, such as diminution of cell viability and cell death. As used herein, "dendrogenesis" refers to the formation and growth of dendrites that extend from the cell body or soma of a neuron. Growth of dendrites includes, but is not limited to processes such as branching, elongation and formation of spines. Embodiments of the present invention provide methods of treating cancer in a subject with at least one antineoplastic compound, the improvement comprises administering to the subject an activator or inhibitor of a Rho family signaling pathway protein in an amount effective to inhibit neuronal impairment in the subject caused by the antineoplastic compound. In some embodiments, the subject is administered an inhibitor of a Rho family signaling pathway protein. In other embodiments, the subject is administered an activator of a Rho family signaling pathway protein.

1. Rho Family Signaling Pathway Proteins Rho family signaling pathway proteins include Rho proteins that are involved in the Rho GTPase signal transduction pathway and includes proteins involved downstream. Rho family signaling pathway proteins include, but are not limited to, Rho GTPases, such as RhoA, RhoB, RhoC, Rac, and Cdc42 subfamily; Rho GTPase activators, such as GEF or guanine nucleotide exchange factors); Rho GTPase inactivators, such as Rho GAP (GTPase activating protein); Rho GTPase GDIs (guanine nucleotide dissociation inhibitors); Rho family GTPase downstream effectors, such as Rho kinase (plόOROCK) and PAK (p21 -activated kinase). Other examples of Rho family signaling pathway proteins can be found in Burridge et al. Cell 116:167-179 (2004) and Mackay et al. J Biol. Chem. 273(33):20685-20688 (1998). In particular embodiments of the present invention, the Rho family signaling pathway protein is a Rho kinase or a Rho GTPase. In some embodiments, the inhibitor of the Rho family signaling pathway protein is Rho kinase inhibitor, Y27632, or an analog thereof. See U.S. Patent No. 6,218,410 to Uehata et al. In still other embodiments, the Rho family signaling pathway protein is a Rho kinase inhibitor comprising an amide compound of the formula (I): (I) O Rb Ra C II N I Re wherein Ra is a group of the formula:

(a)

formulas (a) and (b), R is hydrogen, alkyl or cycloalkyl, cycloaalkyl, phenyl or aracyl, which optionally have a substituent on the ring, or a group of the formula:

wherein R⁶ is hydrogen, alkyl or formula: -NR⁸NR⁹ wherein R⁸ and R⁹ are the same or different and each is hydrogen, alkyl, aralkyl or phenyl, R⁷ is hydrogen, alkyl, aralkyl, phenyl, nitro or cyano, or R⁶ and R⁷ in combination show a group forming a heterocycle optionally having, in the ring, oxygen atom, sulfur atom or optionally substituted nitrogen atom,

R¹ is hydrogen, alkyl or cycloalkyl, cycloalkylalkyl, phenyl or aralky, which optionally have a substituent on the ring, or R and R¹ in combination form, together with the adjacent nitrogen atom, a group forming a heterocycle optionally having, in the ring, oxygen atom, sulfur atom or optionally substituted nitrogen atom,

R is hydrogen or alkyl,

R³ and R⁴ are the same or different and each is hydrogen, alkyl, aralkyl, halogen, nitro, amino, alkylamino, acylamino, hydroxy, alkoxy, aralkyloxy, cyano, acyl, mercapto, alkylthio, aralkylthio, carboxy, alkoxycarbonyl, carbamoyl, alkylcarbamoyl or azide, and

A is a group of the formula: _Rιo -(CH₂)l(C)m(CH₂)n- R¹¹ wherein R¹⁰ and R¹¹ are the same or different and each is hydrogen, alkyl, haloalkyl, aralkyl, hydroxyalkyl, carboxy or alkoxycarbonyl, or R¹⁰ and R¹¹ show a group which forms cycloalkyl in combination and 1, m and n are each 0 or an integer of 1-3, Rb is a hydrogen, an alkyl, an aralkyl, an aminoalkyl or a mono or dialkylaminoalkyl; and Re is an optionally substituted pyridine, triazine, pyrimidine, pyrrolopyridine, pyrazolopyridine, pyrazolopyrimidine, 2,3- dihydropyrrolopyridine, imidazopyridine, pyrrolopyrimidine, imindazopyrimidine, pyrrolotriazine, pyrazolotriazine, triazolopyridine, triazolopyrimidine, or 2,3- dihydropyrrolopyrimidine, an isomer thereof and/or a pharmaceutically acceptable acid addition salt thereof.

2. Antineoplastic Compounds Antineoplastic compounds according to embodiments of the present invention include, but are not limited to, warfarin, heparin, minocycline and drugs which have an antineoplastic effect that are currently in Phase I, II and/or III trials; epothilones, analogs of epothilones, and their class of compounds; melphalan carmustine, busulfan, lomustine, cyclophosphamide, dacarbazine, polifeprosan 20 with carmustine implant, sterile ifosfamide, chlorambucil, mechlorethamine, busulfan, cyclophosphamide, carboplatin, cisplatin, thiotepa, capecitabine, streptozocin, bicalutamide, flutamide, leuprohde acetate, nilutamide, leuprohde acetate, doxorubicin hydrochloride, bleomycin sulfate, daunorubicin hydrochloride, dactinomycin, daunorubicin citrate liposome injection, doxorubicin hydrochloride liposome injection, epirubicin hydrochloride, idarubicin hydrochloride, mitomycin doxorubicin, valrubicin, anastrozole, toremifene citrate, fluorouracil, cytarabine, fluorouracil, fludarabine, floxuridine, interferon alfa-2b, recombinant, plicamycin, mercaptopurine, methotrexate, interferon alfa-2a, recombinant, medroxyprogersterone acetate, estramustine phosphate sodium, estradiol, leuprohde acetate, megestrol acetate, octreotide acetate, octreotide acetate for injection suspension, deithylstilbestrol diphosphate, testolactone, goserelin acetate, etoposide phosphate, vincristine sulfate, etoposide, vinblastine, etoposide, vincristine sulfate, teniposide, trastuzumab, gemtuzumab ozogamicin, rituximab, exemestane, irinotecan hydrocholride, asparaginase, gemcitabine hydrochloride, altretamine, topotecan hydrochloride, hydroxyurea, cladribine, mitotane, procarbazine hydrochloride, vinorelbine tartrate, pentrostatin sodium, mitoxantrone, pegaspargase, denileukin diftitix, altretinoin, porfimer, bexarotene, paclitaxel, docetaxel, temozolomide, bacillus of Calmette and Guerin, arsenic trioxide, tretinoin as listed in U.S. Patent No. 6,581,606 to Kutzko et al. In some embodiments, the antineoplastic compound is imatinib, cisplatin, methotrexate or analogs thereof, and pharmaceutically acceptable salts thereof. In particular embodiments, the antineoplastic compound is a compound of the formula (II): (II)

wherein R and R are the same or different, and respectively represent: hydrogen, C_MO alkyl, C₂.₅ alkanoyl, formyl, C alkoxy-carbonyl, amidino, C₃.₇ cycloalkyl, C₃.₇ cycloalkyl-carbonyl, unsubstituted or substituted phenyl, phenylalkyl, benzoyl, naphthoyl, phenylalkoxy-carbonyl, pyridylcarbonyl or piperidyl, wherein the substituent is selected from the group consisting of halogen, C alkyl, C_M alkoxy, phenylalkyl, nitro or amino, R and R together form unsubstituted or substituted benzylidene, pyrrolidylidene or piperidylidene, wherein the substituent is selected from the group consisting of halogen, C_M alkyl, C alkoxy, phenylalkyl, nitro or amino, or R and R together with the adjacent nitrogen atom form pyrrolidinyl, piperidino, piperazinyl, morpholino, thiomorpholino or phthalimido, R³ represents hydrogen or C alkyl, R⁴ represents a hydrogen or C_M alkyl, R⁵ represents hydrogen, hydroxy, C_M alkyl or phenylalkoxy, R⁶ represents hydrogen or C_M alkyl, A represents single bond, Cι-₅ straight chain alkylene, or alkylene which is substituted by CM alkyl and n represents 0 to 1 , and an optical isomer thereof or a pharmaceutically acceptable acid addition sal thereof.

A. Imatinib In some embodiments of the present invention the antineoplastic compound is imatinib or a pharmaceutically acceptable salt thereof such as ST1571. See U.S. Patent No. 4,997,834 to Muro et al. STI571 is a small molecule inhibitor of Abl family tyrosine kinases. STI571 is commercially available as Gleevec® (imatinib mesylate) from Novartis International AG as film-coated tablets containing imatinib mesylate equivalent to 100 mg or 400 mg of imatinib free base. In particular embodiments, imatinib or an analog thereof or a pharmaceutically acceptable salt thereof is administered orally. Dosages can be determined in accordance with routine pharmacological procedures known to those skilled in the art. In some embodiments, imatinib or a pharmaceutically acceptable salt thereof is administered in a range from about 50 mg to about lg per day. The frequency of administration is usually one, two, or three times per day or as necessary to control the condition. The duration of treatment depends on the type and severity of the condition being treated and the type and age of the subject being treated. In particular embodiments, the present invention provides methods of treating cancer in a subject with imatinib or an analog thereof or a pharmaceutically acceptable salt thereof, the improvement comprises administering to the subject a Rho kinase inhibitor in an amount effective to enhance the antineoplastic activity or reduce the neurotoxicity of the imatinib or analog thereof or a pharmaceutically acceptable salt thereof. In some embodiments of the present invention, administering to the subject a Rho kinase inhibitor in an amount effective to enhance the antineoplastic activity or reduce the neurotoxicity of the imatinib or analog thereof or a pharmaceutically acceptable salt thereof may result in improved patient mental function, reduced anxiety, reduced depression, positive immune responses, physical well-being and combinations thereof. In some embodiments, improved patient mental function can be improvements in some or all of the following areas: attention span, concentration, memory, visual ability, verbal ability, mental flexibility, mental processing speed and motor speed. In particular embodiments, the Rho kinase inhibitor is Y27632, or an analog thereof. B. Cisplatin In some embodiments, the antineoplastic agent is cisplatin. Cisplatin is an alkylating agent that has a heavy metal complex containing a central atom of platinum surrounded by two chloride atoms and two ammonia molecules in the cis position. In particular embodiments, cisplatin is administered parenterally. Dosages can be determined in accordance with routine pharmacological procedures known to those skilled in the art. In some embodiments, cisplatin is administered in a range from about 10 mg/m² to about 200 mg/m² IN for a period deemed sufficient in view of the condition being treated. Thus, the frequency of administration is usually one, two, or three times per day or as necessary to control the condition. The duration of treatment depends on the type and severity of the condition being treated and the type and age of the subject being treated.

C. Methotrexate In some embodiments, the antineoplastic agent is methotrexate. Methotrexate is a folate antagonist. In some embodiments, methotrexate is administered orally or parenterally. Dosages can be determined in accordance with routine pharmacological procedures known to those skilled in the art. In some embodiments, methotrexate is administered in a range from about 2.5 mg to about 50 mg for a period deemed sufficient in view of the condition being treated. Thus, the frequency of administration is usually one, two, or three times per day or as necessary to control the condition. The duration of treatment depends on the type and severity of the condition being treated and the type and age of the subject being treated. In particular embodiments of the present invention, the antineoplastic compound is toxic to neurons. 3. Formulations and Dosages

The compounds described above can be formulated for administration in accordance with known pharmacy techniques. See, e.g., Remington, The Science And Practice of Pharmacy (9th ed. 1995). In the manufacture of a pharmaceutical composition according to the present invention, the active compound (including the isomers and physiologically acceptable salts thereof) is typically admixed with, ter alia, an acceptable carrier. The carrier must, of course, be acceptable in the sense of being compatible with any other ingredients in the formulation and must not be deleterious to the patient. The carrier can be a solid or a liquid, or both, and is preferably formulated with the compound as a unit-dose formulation. One or more active compounds can be incorporated in the compositions of the invention, which can be prepared by any of the well-known techniques of pharmacy. Methods according to embodiments of the present invention provide administration of antineoplastic compounds and activators or inhibitors of a Rho family signaling pathway proteins. Administration of these compounds may be oral, rectal, topical, inhalation (e.g., via an aerosol) buccal (e.g., sub-lingual), vaginal, topical (i.e., both skin and mucosal surfaces, including airway surfaces), transdermal administration and parenteral (e.g., subcutaneous, intramuscular, intradermal, intraarticular, intrapleural, intraperitoneal, intracerebral, intraarterial, or intravenous), although the most suitable route in any given case will depend on the nature and severity of the condition being treated and on the nature of the particular active agent which is being used. Parenteral formulations can comprise the active compound, which preparations are preferably isotonic with the blood of the intended recipient. These preparations can be administered by means of subcutaneous, intramuscular, intradermal, intrathecal injection, epidural injection, intraventricular injection into a ventricle of the brain or intravenous injection. Such preparations may conveniently be prepared by admixing the compound with an agent to render the resulting solution sterile and isotonic with the blood. In addition to active agents or their salts, the pharmaceutical compositions can contain other additives, such as pH-adjusting additives. In particular, useful pH- adjusting agents include acids, such as hydrochloric acid, bases or buffers, such as sodium lactate, sodium acetate, sodium phosphate, sodium citrate, sodium borate, or sodium gluconate. Further, the compositions can contain microbial preservatives. Useful microbial preservatives include methylparaben, propylparaben, and benzyl alcohol. The microbial preservative is typically employed when the formulation is placed in a vial designed for multidose use. The pharmaceutical compositions of the present invention can be lyophilized using techniques well known in the art.

4. Treatment and Administration Embodiments of the present invention provide treatment of hyperproliferative disorders such as cancers, tumors and neoplastic disorders, as well as premalignant and non-neoplastic or non-malignant hyperproliferative disorders. Examples of tumors, cancers, and neoplastic tissue that can be treated by the present invention include, but are not limited to, malignant disorders such as breast cancers; osteosarcomas; angiosarcomas; fibrosarcomas and other sarcomas; leukemias; lymphomas; sinus tumors; ovarian, uretal, bladder, prostate and other genitourinary cancers; colon, esophageal and stomach cancers and other gastrointestinal cancers; lung cancers; myelomas; pancreatic cancers; liver cancers; kidney cancers; endocrine cancers; skin cancers; and brain or central and peripheral nervous (CNS) system tumors, malignant or benign, including gliomas and neuroblastomas. Examples of premalignant and non-neoplastic or non-malignant hyperproliferative disorders include, but are not limited to, myelodysplastic disorders; cervical carcinoma-in-situ; familial intestinal polyposes such as Gardner syndrome; oral leukoplakias; histiocytoses; keloids; hemangiomas; hyperproliferative arterial stenosis, inflammatory arthritis; hyperkeratoses and papulosquamous eruptions including arthritis. Also included are viral induced hyperproliferative diseases such as warts and EBN induced disease (i.e., infectious mononucleosis), scar formation, and the like. The methods of treatment disclosed herein may be employed with any subject known or suspected of carrying or at risk of developing a hyperproliferative disorder as defined herein. Subjects suitable to be treated according to the present invention include any mammalian subject in need of being treated according to the present invention. Human subjects are preferred. Human subjects of both genders and at any stage of development (i.e., neonate, infant, juvenile, adolescent, adult) can be treated according to the present invention. Mammalian subjects according to embodiments of the present invention include, but are not limited to, canines, felines, bovines, caprines, equines, ovines, porcines, rodents (e.g. rats and mice), lagomorphs, primates, humans, and the like, and mammals in utero. The present invention is primarily concerned with the treatment of human subjects, but the invention can also be carried out on animal subjects, particularly mammalian subjects such as mice, rats, dogs, cats, livestock and horses for veterinary purposes, and for drug screening and drug development purposes. As used herein, "treatment" of a cancer refers to methods of killing, inhibiting or slowing the growth or increase in size of a body or population of hyperproliferative cells or tumor or cancerous growth, reducing hyperproliferative cell numbers, or preventing spread to other anatomic sites, as well as reducing the size of a hyperproliferative growth or numbers of hyperproliferative cells. As used herein, "treatment" is not necessarily meant to imply cure or complete abolition of hyperproliferative growths. As used herein, a "treatment effective amount" is an amount effective to result in the killing, the slowing of the rate of growth of hyperproliferative cells or tumor or cancerous growth, the decrease in size of a body of hyperproliferative cells or tumor or cancerous growth, and/or the reduction in number of hyperproliferative cells or tumor or cancerous growth. The treatment effective amount of the active agent, the use of which is in the scope of present invention, will vary somewhat from patient to patient, and will depend upon factors such as the age and condition of the patient and the route of delivery. A potentiating agent (or agents) can be included in an amount sufficient to enhance the activity of the first compound, such that the two (or more) compounds together have greater therapeutic efficacy than the individual compounds given alone (e.g., due to synergistic interaction; reduced combined toxicity, etc.). Dosages can be determined in accordance with routine pharmacological procedures known to those skilled in the art. As a general proposition, a dosage from about 0.1 to about 50 mg kg/day will have therapeutic efficacy, with all weights being calculated based upon the weight of the active agent. Toxicity concerns at the higher level may restrict dosage. A dosage from about 10 mg/kg to about 50 mg/kg may be employed for oral administration. The frequency of administration is usually one, two, or three times per day as a bolus, or by intravenous infusion, or as necessary to control the condition. The duration of treatment depends on the type of condition being treated and may be for as long as the life of the patient. In particular embodiments, the active compounds can be administered in combination. As used herein, the administration of two or more compounds "in combination" means that the at least two compounds are administered closely enough in time that the presence of one alters the biological effects of the other. The at least two compounds can be administered simultaneously (concurrently) or sequentially. Simultaneous administration can be carried out by mixing the compounds prior to administration, or by administering the compounds at the same point in time but at different anatomic sites or using different routes of administration. The phrases "concurrent administration", "administration in combination", "simultaneous administration" or "administered simultaneously" as used herein, means that the compounds are administered at the same point in time or immediately following one another. In the latter case, the two compounds are administered at times sufficiently close that the results observed are indistinguishable from those achieved when the compounds are administered at the same point in time.

5. Screening Methods The present invention further provides methods of screening for compounds useful for inhibiting neuronal impairment caused by administering an antineoplastic compound comprising contacting a neuronal cell with a test compound, in the presence of an Abl family tyrosine kinase inhibitor, wherein the contacting step is carried out in vitro; and then determining whether the test compound affects dendrogenesis influenced by Abl family tyrosine kinase inhibition; a positive effect on the dendrogenesis influenced by the Abl family tyrosine kinase inhibition indicates that the test compound is useful for inhibiting neuronal impairment caused by administering an antineoplastic compound. In some embodiments, the Abl family tyrosine kinase inhibitors can inhibit Abl family tyrosine kinases such as c-Abl (Abl), BCR-Abl, v-Abl, Arg (Abl-related gene), and Drosophila Abl (D-Abl). In other embodiments, the Abl family tyrosine kinase inhibitor is imatinib or an analog thereof or a pharmaceutically acceptable salt thereof. In still other embodiments, imatinib or an analog thereof or a pharmaceutically acceptable salt thereof can inhibit Abl family tyrosine kinases, platelet derived growth factor (PDGF) receptor tyrosine kinase or Kit family tyrosine kinases. Further embodiments of the present invention will now be described with reference to the following examples. It should be appreciated that these examples are for the purposes of illustrating embodiments of the present invention, and do not limit the scope of the invention.

EXAMPLE 1 Material and Methods

Materials Full length δ-catenin was subcloned into pEGFP-C2 as described by Lu et al., (1999). To create the C-terminal deletion construct ΔC207, the full-length δ-catenin from pEGFP-δ-catenin was excised with the use of the restriction enzymes Nru I and Xma I, blunt-ended and ligated in order. After plasmid construction, the sequences of δ-catenin constructs in pEGFP-C2 were confirmed by DNA sequencing and found to be in frame. Hippocampal cultures and pharmacological treatment Embryonic hippocampal cultures were prepared as described by Goslin and Banker (1998) with minor modifications (Lu et al., 1999; Jones et al., 2002). Briefly, 18-day timed pregnant rats were sacrificed and the embryos were removed in accordance with the NIH Guide for the Care and Use of Laboratory Animals. Hippocampi were collected and cells are dissociated by trypsinization and plated onto poly-L-lysine coated coverslips. After neurons adhered to the substrate, the medium was changed over to B-27 supplemented Neurobasil. This culture scheme allowed us to maintain viable low-density hippocampal cultures for several weeks.

Time-lapse light microscopy To determine the dynamics of dendrogenesis of a single neuron, we have applied time-lapse light microscopy according to a procedure described earlier (Jones et al., 2002; Kim et al., 2002). Briefly, dissociated hippocampal neurons treated with DMSO as control or STI571 for two days from 5DIV to 7DIV were grown on 25 mm coverslips and placed in an Attofluor cell chamber (Atto Instruments, Rockville, MD) on the heated stage of Zeiss Axiovert microscope, which is supported by an anti- vibration table. Differential interference contrast images at 63x were captured using a Hamamatsu Orca camera. To minimize phototoxicity for the living cells, a computer driven, automatic shutter was used to achieve minimum illumination (300 millisecond per frame). The longest continuous recording was 135 minutes. For some experiments, at the end of time-lapse experiments, cells were fixed and processed for Hoechst 33258 nuclear staining and anti-MAP2 immunocytochemistry to evaluate the status of neuronal cell death. The integrity of nuclear and cell morphology as well as dendritic motility indicated that neurons were healthy throughout the incubation period. For some time-lapse experiments, neurons were first grown in normal medium for 7 days and then were placed on stage for recording. DMSO or STI571 was added for 135 minutes to examine the on-set of dendritic deterioration caused by STI571. Recorded time-series were analyzed using MetaMorph 4.6 (Universal Imaging Inc, West Chester, PA). Dendrite length is determined using REGION MEASUREMENT function and numerical values of length measurement are logged onto Microsoft Excel automatically. All length measurement was calibrated so that the length reflected true values. The histories of their elongation and retraction are produced using MS Excel and SigmaPlot 5.0 (SPSS Science). Student t tests were performed and ^-values were assigned in each experiment. Any null hypothesis with the probability level less than 99 % was rejected.

DNA transfection and immunofluorescent light microscopy Hippocampal neurons grown in culture for 5 days were transfected with different pEGFP δ-catenin cDNAs by using the modified calcium precipitation method (Xia et al., 1996; Goslin et al., 1998). The neurons were fixed in 4 % paraformaldehyde 48 hours after the transfection. Alternatively, transfected neurons were subject to either 5 minutes of detergent extraction (1% Triton X-100 in 60 mM

PIPES buffer containing 25 mM HEPES, 10 mM EGTA and 2 mM MgSO₄ and 4 % PEG 20,000) or 5 μM Latrunculin A (Biomol Research Laboratory, Inc. Plymouth

Meeting, PA) treatment for 24 hours before they were fixed for immunofluorescent light microscopy. For double labeling experiments, neurons were treated with 0.2 %

Triton X-100 for 15 minutes, blocked with 10 % BSA, and stained with rhodamine phalloidin (Molecular Probes, Inc, Eugene, OR). After PBS washes, the coverslips were mounted and analyzed under either a Zeiss Axiovert SI 00 or a Zeiss LSM 510 confocal laser scanning light microscope (Carl Zeiss, Thornwood, NY). The morphometric analyses were performed using MetaMorph 4.6 Imaging software system (Universal Imaging Corp. West Chester, PA). Unless otherwise indicated, all chemicals were from Sigma Chemical Co (St. Louis, MO).

Protein immunoprecipitation and Immunoblotting The dissociated embryonic neurons cultured in vitro for 21 days were either lysed in a non-denaturing immunoprecipitation (IP) buffer that contains 10 mM Tris, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1 % Triton X-100 and 0.5 % NP- 40 with protease and phosphatase inhibitor cocktails or in a denaturing buffer containing the above reagents and 0.2% SDS. For brain protein immunoprecipitation, adult rat brains were homogenized in 10 mM Tris, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 0.1 % Triton X-100 and 0.1 % NP-40 containing protease and phosphatase inhibitor cocktails. The cell debris and particulates were removed by centrifugation at 5000 g for 30 minutes. The supernatants were centrifuged at 100,000 g for 60 minutes. The insoluble pellets were collected and homogenized in IP buffer. The insoluble materials were removed by centrifugation at 100,000 g for 60 minutes. After preclearing with protein A-Sepharose beads, the lysates were either mixed with sample buffer for SDS-PAGE analysis, or for further immunoprecipitation experiments. The precleared lysates were adjusted to 2 mg total protein and were immunoprecipitated with specific antibodies, and the immunoprecipitates were captured by protein A-Sepharose (or protein G plus protein A-agarose beads for the immunoprecipitations using mouse monoclonal antibodies). Samples were washed three times with the IP buffer. The immunoprecipitates as well as 20 μg lysates per well were separated on a SDS-PAGE and transferred to nitrocellulose membranes (PGC Scientifics, Frederick, MD). For some experiments, 100 μM L-glutamate or 100 μM NMDA was prepared in the buffer containing DMEM with 10 mM glycine and 0.2 g/Liter Na₂HPO4, pH 7.2 and added to the culture for 5, 10 or 60 minutes. The cells were then lysed and the immunoprecipitations were performed as described above. Proteins co-immunoprecipitated with δ-catenin were revealed by anti-NR2A, PSD95, mGluRl, or δ-catenin immunoblotting using enhanced chemiluminescence (Amersham Life Science, Piscataway, NJ) for detection. When necessary, blots were stripped with 100 mM β-mercaptoethanol, 2% SDS, and 62.5 mM Tris-HCl, pH 7.6 for 30 min at 55 ° C. The blots were then washed in Tris buffer containing 0.1% Tween 20 before re-probing with specific antibodies. EXAMPLE 2 Inhibition of Abl family tyrosine kinase activity disrupts dendrogenesis independent of axonogenesis Hippocampal neurons acquired their polarized morphology in a well- characterized sequence of events (Dotti et al., 1988). In order to examine what role the Abl family kinases may play during dendrogenesis, we compared the developmental progression of primary hippocampal neurons to those cultures treated with STI571 (Or CGP15748/Gleevac/Imatinib Mesylate), a well-known small molecule inhibitor of Abl family kinase activity (Buchdunger et al., 1996; Carroll et al., 1997; Lu et al., 2002). In control cells, typical formation of lamellipodial structures from the spherical cell body occurs by 0.5 DIN (Days In Nitro)(Figure 1 A). This is followed by the extension of multiple neurites, one of which assumes axonal identity, by 3 DIN. Between this point and 7 DIN, dendritic identity is established and dendritic processes elongate and become more structurally complex, while axonal outgrowth continues independently. In order to examine the effects of Abl family kinase inhibition during dendrogenseis, STI571 was applied to hippocampal neuron cultures between 4.5 DIN and 7 DIN. This allowed us to examine the effects of Abl family kinase inhibition on dendrogenesis after the axonal identity has been fully established. After 24 hours (at 5 DIN), STI571 treatment resulted in a reduction of dendrite length as well as a corresponding attenuation of branch formation (Figure IB, arrows point to dendrites while arrowheads point to the axon). This trend continued through 6 DIN, when dendrite length and branch formation were both markedly decreased. By 7 DIN, dendritic development was completely inhibited and their degeneration was apparent. The effects of STI571 on dendrogensis were dose dependent, as 60 nM had little impact while maximum inhibition was achieved at 5 μM treatment. In addition, STI571 effects were reversible, since after its withdrawal on 7 DIN, dendrogenesis resumed and showed no significant difference by 10 DIN when compared to control neurons. We noted that Abl kinase inhibition had a less striking effect upon axon morphology after axons established their identity. Indeed, axons in both treatments continued to grow and form branches at similar rates (Figure 1A and IB). These data indicate that Abl family kinases play an important role in the formation of dendritic arbor, and that this involvement may be separable from axonogenesis. EXAMPLE 3 Inhibition of Abl family tyrosine kinase activity alters the developmental dynamics of dendrogenesis To examine the dynamics of dendrogenesis influenced by Abl kinase inhibition, we employed time-lapse microscopic imaging method. Control neurons treated with DMSO at 7 DIN displayed rapid motility of primary and secondary dendrites, as well as extension and retraction of dendritic sprouts. We designated the primary dendrites to those processes longer than 10 μm and they were extended directly from the neuronal cell body. The secondary dendrites were those that branched off directly the primary dendrites. At the end of a 135-minutes recording experiment, primary dendrites increased length significantly (Fig 2A. Asterisks point to the extension of one such dendrite). During this time period, an individual dendrite can elongate and retract for a few minutes, followed by a major advance (Fig 2B). In one example of examining the average life history of secondary dendrites, two major advances can be demonstrated, leading to a 45 % increase in length at the end of the recording period (Fig 2B, DMSO). At 7DIN, primary dendrite extended at a slower rate, giving a 20 % increase in length at the end of the recording period (Fig 2C, DMSO). In contrast, while individual secondary dendrites in neurons treated with STI571 from 5 DIN to 7 DIN could show a modest elongation (Fig 2 A arrowhead), the length of most other dendrites remained unchanged or was slightly decreased (Fig 2A, # points to a retracting dendrite). This resulted in no overall elongation of primary dendrites and an average of 10 % increase in secondary dendrite length but that is statistically insignificant (Fig 2B and C). In addition, although the dendritic sprout numbers remained unchanged in both control and STI571 treated neurons before and after recording, there were significantly greater numbers of sprout formation in control neurons (Fig 2A and C). While sprout formation did occur, further extension was limited, leading to a net retraction and subsequent reduction in dendrite complexity when neurons were examined two days after STI571 treatment (Fig 2 A). As with previous experiments, no significant differences in axonal outgrowth or growth cone dynamics were noted in response to Abl kinase inhibition following axonal establishment (See supplemental information of time-lapse light microscopy). The effects of STI571 on dendrogenesis seemed rapid, since time-lapse analysis indicated that dendrogenesis paused following 1 hour STI571 treatment at 7 DIN. These experiments suggest that Abl kinase inhibition may impair the function of molecular machinery that is required for dendritic branching that is highly active at 7DIN.

EXAMPLE 4 Inhibition of Abl family tyrosine kinase disrupts axon-dendrite polarity To determine if the cytoskeletal organization of hippocampal neurons is altered by the inhibition of Abl tyrosine kinases, double immunofluorescent light microscopy was performed. At 7 DIN, control neurons showed elaborated dendritic arbor that was supported by the underlying actin network (Fig 3 A, a, arrows). Microtubule-associated protein 2 (MAP-2), specific for dendrite localization, was clearly present in the dendrites (Fig 3A, b, arrows) but excluded from the axon (Fig 3A, b, arrowhead). Microtubule-associated protein tau, known to segregate into the axonal compartment, was present in the axon (Fig 3A, c, arrowhead) but only weakly visible in the dendrites (Fig 3 A, c, arrows). However, while neurons treated with STI571 for 48 hours showed localized enrichment of actin filament at the growth cones of axon (Fig 3B, a, asterisk), weak actin staining in the degenerating dendrites was observed (Fig 3B,a, arrows). A weak anti-MAP-2 immunostaining was seen in the dendrites, but it also became positive in the axon, indicating the failure of MAP-2 segregation (Fig 3B, b). The disruption of normal axon-dendrite polarity was evident, since tau protein was not only present in the axon (Fig 3B, c, arrowheads), but was also observed in the dendrites (Fig 3B, c, arrows).

EXAMPLE 5 Abl family tyrosine kinase activity influences primary dendrite formation and secondary dendritic branching In order to further examine the effect of Abl kinase activity on morphological features of dendrite formation, we quantified characteristics such as primary dendrite formation and secondary dendritic branching in response to Abl kinase inhibition and overexpression of a constitutively active Abl (CA-Abl) (Figure 3). Immunofluorescent light microscopy showed that the endogenous Abl expression was present throughout the neuronal cell body and the processes (Fig 3A, DMSO and STI571), although the immunostaining was rather weak when compared with the intensive Abl immunoreactivity of the cells transfected with CA-Abl (Fig 3 A, CA- Abl). STI571 treatment reduced the numbers of MAP2 positive dendrites when compared to control DMSO treated neuron (Fig 3A, DMSO and STI571), but CA-Abl overexpression induced an exuberant formation of MAP2 positive dendrites (Fig 3 A, CA-Abl). To quantify the numbers of dendrites per neuron and measure the length of dendrites, we applied the REGION FUNCTION of MetaMorph imaging software. Dendrites within the area of 120 μm² were scored. When compared to control, which had an average of 11 primary dendrites and 8 secondary dendrites per cell, Abl inhibitor treated neurons showed only 7 primary dendrites and 4 secondary dendrites per cell (Fig 3B). This is in contrast to the neurons overexpressing CA-Abl, in which a formation of 14 primary dendrites can be observed (Fig 3B). Interestingly, the effects of CA-Abl overexpression appeared to influence primary dendrites more dramatically, since the number of secondary dendrites in these neurons did not increase significantly when compared to control neurons (Fig 3B). We also examined if Abl kinase activity affects the length of primary and secondary dendrites. Control neurons showed an average length of 46 μm for primary dendrites and 23 μm for secondary dendrite, respectively. STI571 treated neurons, however, showed an average primary dendrite length of 38 μm and secondary dendrite length of 18 μm. CA-Abl overexpression led to an increase in primary dendrite length to 55 μm, but had little effect on secondary dendrite length. It is important to note that the primary dendrite formation was so extensive in the CA-Abl transfected neurons that it was sometimes difficult to obtain an accurate measurement for the number of secondary dendrites (Fig 3 A,CA-Abl). Therefore, the number of secondary dendrites in CA-Abl transfected neurons may be underestimated. These analyses demonstrated that a balanced Abl tyrosine kinase activity is important in maintaining a physiological pace of dendrogenesis. EXAMPLE 6 Inhibition of Abl family tyrosine kinase activity leads to an increase in GTP- bound RhoA without affecting Racl activity The morphological features observed upon modulation of Abl kinase activity are reminiscent of those induced by the Rho family of small GTPases, which are important regulators of many aspects of neurogenesis, including dendrogenesis. RhoA has been widely reported to negatively regulate dendrite outgrowth (Nakayama et al., 2000). In order to determine whether the activity of Rho family small GTPases are affected by inhibition of Abl kinase activity, we utilized an affinity precipitation technique to quantitate active (GTP-bound) Rho GTPases (Benard et al., 1999; Ren et al., 1999). Cultured primary neurons were subject to a series of pharmacological treatments to enhance or inhibit Rho family small GTPase activity. When neurons 5DIN were treated with DMSO as control for 48 hours, a basal level of GTP-bound, active RhoA and Racl can be detected (Fig 5, DMSO). STI571 treatment increased RhoA activity, but did not induce significant changes in Racl activity (Fig 5, STI571). Toxin B, a well-established inhibitor of Rho GTPase activity, suppressed GTP-bound RhoA and Racl (Fig 5, Toxin B). We treated neurons with STI571 in the presence of hydrogen peroxide as a positive control, since it further enhanced RhoA activity (Fig 5, STI571+H₂O₂). Glutamate treatment leads to actin remodeling in the dendritic spines and a redistribution of dendrite specific protein such as δ-catenin (Fisher et al., 2000; Jones et al., 2002; Portera-Cailliau et al., 2003). We therefore subjected neurons at 7DIN a brief treatment of glutamate for 10 minutes. Compared to DMSO or STI571 treatment neurons, glutamate induced an activation of Racl (Fig 5, Glutamate). This indicates that RhoA may be a downstream target of Abl kinase activity, while Racl may not be directly targeted.

EXAMPLE 7 Rho Kinase inhibition suppresses STI571 effect on dendrogenesis While RhoA has many downstream effectors, Rho associated kinase or pl60/ROCK, has been shown to be particularly important in neuronal morphogenesis. This is demonstrated in several studies that have shown that RhoA mediated process retraction can be prevented by inhibition of ROCK activity (Hirose et al., 1998; Bito et al., 2000; Nakayama et al., 2000). Since morphological and biochemical data suggest an association of the Abl family kinases and RhoA signaling, we speculated that ROCK may also lie downstream of Abl. To examine this more closely, we used Y-27632, a ROCK specific inhibitor (Uehata et al., 1997), to determine whether the STI571 induced dendritic simplification can by reversed when ROCK activity is blocked. Double fluorescent light microscopy showed that neurons 7DIN showed an elaborate dendrite formation that was MAP2 positive, and displayed a well-defined F- actin network (Fig 6A, STI571 : - and Y27632: -). STI571 treatment suppressed dendritic elaboration that was represented by a reduced number of MAP2 positive dendrites, although the fluorescent phalloidin staining showed that F-actin intensity

94 was not reduced (Fig 6A, STI571 : + and Y27632: -). Neurons treated with Y27632 alone promoted the formation of MAP2 positive dendrites, and also apparently an increased dendrite length (Fig 6A, STI571: - and Y27632: +). When compared to STI571 treatment alone, STI571 in the presence of Y27632 elicited a reversal of dendritic simplification back to the level of arborization seen in control cells (Fig 6A, STI571 : + and Y27632: +). The increase in dendrite complexity upon treatment with Y-27632 alone is consistent with a previous report demonstrating a role for ROCK inhibition in the formation of multiple neurites with increased length in the initial phase of neuritogenesis (Da Silva et al., 2003). When the effects of Y27632 on dendrogenesis were quantified, it became clear that, while STI571 inhibited both the numbers of primary and secondary dendrites, Y27632 was able to suppress the effect of STI571 (Fig 6B). Y27632 treatment alone did not significantly affect the number of dendrites. STI571 also reduced the length of both primary and secondary dendrites when compared to neurons treated with DMSO as control (Fig 6C). Y27632 treatment alone, however, increased the dendrite length dramatically. Neurons treated with Y27632 reversed the inhibitory effect of STI571 on the length of secondary dendrites, but further promoted the length of primary dendrites when compared to neurons treated with DMSO. The ability of Y27632 to reverse STI571 effects on dendrites was rather extensive, since Y27632 was applied to neurons already treated with STI571 for 24 hours. Thus, our results indicate that Abl kinase activity indeed plays a critical role in the upstream regulation of both RhoA and it's downstream effector, ROCK during dendrogenesis.

EXAMPLE 8 RhoA activation suppresses CA-Abl effect on dendrogenesis In addition to the simplification of the dendritic tree that was observed upon inhibition of the Abl family kinases, we also found a reciprocal increase in dendritic complexity upon overexpression of a constitutively active Abl kinase. If Abl kinase activity negatively regulates RhoA, it stands to reason that expression of constitutively active RhoA may be sufficient to block the phenotype seen with Abl kinase activation. In order to test this hypothesis, we co-expressed constitutively active Abl (CA-Abl) and constitutively active RhoA (CA-RhoA) constructs (Fig 7). As shown in Fig 4A (CA-Abl) and Fig 7 (CA-Abl+pEGFP-C2), constitutive activation of Abl kinase induced an exuberant formation of dendritic arbors. As

75 previously reported, CA-RhoA was sufficient to induce dendrite retraction and resulted in a drastic simplification of the dendritic arbor (Fig 7, CA-RhoA-GFP; Nakayama et al., 2000). When CA-RhoA and CA-Abl are co-expressed, we observed a complete reversal of the CA-Abl phenotype to that which was seen with CA-RhoA alone (Fig 7, CA-Abl + CA-RhoA-GFP). Overall, these data indicate that the activity of Abl kinase plays a critical role in the regulation of dendrite elaboration during dendrogenesis through a RhoA/ROCK dependent mechanism.

EXAMPLE 9 The CA-Abl effect on dendrogenesis is dependent upon the reorganization of the actin cytoskeleton but is independent of microtubule stability Modulation of Abl kinase activity appears to signal through RhoA and ROCK, both of which have been shown to be essential in the regulation of actin stability and neurite sprouting (Da Silva et al., 2003). We have speculated that Abl kinase activity may also affect the underlying cytoskeletal structure of primary neurons in association with RhoA/ROCK dependent signaling. To examine this more closely, we pharmacologically modified the actin cytoskeleton in conjunction with overexpression of CA-Abl kinase to determine whether the CA-Abl phenotype is dependent upon reorganization of actin (Fig 8 A). Upon microfilament destabilization with Latrunculin A alone, we noted an increase in the neuronal process formation which resembles the phenotype generated by CA-Abl (Compare F-actin in Fig 8A, CA-Abl and Latrunculin A). Treatment with Latrunculin A in conjunction with CA-Abl overexpression yielded a phenotype similar to both treatments individually, which indicates the requirement for actin disruption in process formation in response to Abl activation (Fig 8A, CA-Abl/+ and Latrunculin A/+). In the reverse experiment, we used jasplakinolide, a cell permeable peptide that is a potent inducer of actin polymerization to determine whether the CA-Abl phenotype can be blocked by stabilization of the actin cytoskeleton. Treatment with jasplakinolide alone resulted in a slight simplification of dendritic branching (Fig 8A, jasplakinolide/+). When CA- Abl expression was combined with jasplakinolide treatment, the extensive branching was partially reversed, providing evidence that Abl may function in a dendritic morphogenesis context by destabilizing the actin cytoskeleton. We also addressed a possible role for microtubule stability in the CA-Abl induced branching phenotype (Fig 8B). When microtubules were depolymerized by Vincristine treatment, neither

Ifx control cell morphology nor the CA-Abl phenotypes were reversed, indicating that stabilized microtubules are not required for the maintenance of either morphology. These results are consistent with the hypothesis that the CA-Abl phenotype is dependent upon the stability of the actin cytoskeleton, but is independent of microtubule stability.

The foregoing is illustrative of the present invention, and is not to be construed as limiting thereof. The invention is defined by the following claims, with equivalents of the claims to be included therein.

Claims

THAT WHICH IS CLAIMED IS:

1. In a method of treating cancer in a subject with at least one antineoplastic compound, the improvement comprising: administering said subject an activator or inhibitor of a Rho family signaling pathway protein in an amount effective to inhibit neuronal impairment in said subject caused by said antineoplastic compound.

2. The method of claim 1, wherein said subject is administered an inhibitor of a Rho family signaling pathway protein.

3. The method of claim 1, wherein said subject is administered an activator of a Rho family signaling pathway protein.

4. The method of claim 1 , wherein said Rho family signaling pathway protein is a Rho kinase or a Rho GTPase.

5. The method of claim 1, wherein the inhibitor of said Rho family signaling pathway protein is Rho kinase inhibitor, Y27632, or an analog thereof.

6. The method of claim 1 , wherein said Rho family signaling pathway protein is a Rho kinase inhibitor comprising an amide compound of the formula (I): (I) O Rb Ra C II N I Re wherein Ra is a group of the formula: (a)

?R

wherein R⁶ is hydrogen, alkyl or formula: -NR⁸NR⁹ wherein R⁸ and R⁹ are the same or different and each is hydrogen, alkyl, aralkyl or phenyl, R⁷ is hydrogen, alkyl, aralkyl, phenyl, nitro or cyano, or R⁶ and R⁷ in combination show a group forming a heterocycle optionally having, in the ring, oxygen atom, sulfur atom or optionally substituted nitrogen atom, R¹ is hydrogen, alkyl or cycloalkyl, cycloalkylalkyl, phenyl or aralky, which optionally have a substituent on the ring, or R and R¹ in combination form, together with the adjacent nitrogen atom, a group forming a heterocycle optionally having, in the ring, oxygen atom, sulfur atom or optionally substituted nitrogen atom, R is hydrogen or alkyl,

R³ and R⁴ are the same or different and each is hydrogen, alkyl, aralkyl, halogen, nitro, amino, alkylamino, acylamino, hydroxy, alkoxy, aralkyloxy, cyano, acyl, mercapto, alkylthio, aralkylthio, carboxy, alkoxycarbonyl, carbamoyl, alkylcarbamoyl or azide, and A is a group of the formula:

99 _R.o -(CH₂)l(C)m(CH₂)n- R" wherein R¹⁰ and R^u are the same or different and each is hydrogen, alkyl, haloalkyl, aralkyl, hydroxyalkyl, carboxy or alkoxycarbonyl, or R¹⁰ and R¹¹ show a group which forms cycloalkyl in combination and 1, m and n are each 0 or an integer of 1-3, Rb is a hydrogen, an alkyl, an aralkyl, an aminoalkyl or a mono or dialkylaminoalkyl; and Re is an optionally substituted pyridine, triazine, pyrimidine, pyrrolopyridine, pyrazolopyridine, pyrazolopyrimidine, 2,3- dihydropyrrolopyridine, imidazopyridine, pyrrolopyrimidine, imindazopyrimidine, pyrrolotriazine, pyrazolotriazine, triazolopyridine, triazolopyrimidine, or 2,3-dihydropyrrolopyrimidine, an isomer thereof and/or a pharmaceutically acceptable acid addition salt thereof.

7. The method of claim 1, wherein said antineoplastic compound is toxic to neurons in said subject.

8. The method of claim 1, wherein said neuronal impairment is selected from the group consisting of neuronal cell death, impaired dendrogenesis, actin- cytoskeletal reorganization and chemotherapy-induced DNA damage.

9. The method of claim 1, wherein said antineoplastic compound is selected from the group consisting of imatinib, cisplatin, pachtaxel, and methotrexate, analogs thereof, and pharmaceutically acceptable salts thereof.

10. The method of claim 1, wherein said antineoplastic compound is a compound of the formula (II): (II)

wherein R and R are the same or different, and respectively represent: hydrogen, C _O alkyl, C₂.₅ alkanoyl, formyl, C_M alkoxy-carbonyl, amidino, C₃.₇ cycloalkyl, C₃- cycloalkyl-carbonyl, unsubstituted or substituted phenyl, phenylalkyl, benzoyl, naphthoyl, phenylalkoxy-carbonyl, pyridylcarbonyl or piperidyl, wherein the substituent is selected from the group consisting of halogen, C_M alkyl, C_M alkoxy, phenylalkyl, nitro or amino, I 9 R and R together form unsubstituted or substituted benzylidene, pyrrolidylidene or piperidylidene, wherein the substituent is selected from the group consisting of halogen, C_M alkyl, C_M alkoxy, phenylalkyl, nitro or amino, or R and R together with the adjacent nitrogen atom form pyrrolidinyl, piperidino, piperazinyl, morpholino, thiomorpholino or phthalimido, R represents hydrogen or C_M alkyl, R⁴ represents a hydrogen or C_M alkyl, R⁵ represents hydrogen, hydroxy, C_M alkyl or phenylalkoxy, R represents hydrogen or C_M alkyl, A represents single bond, Cι-₅ straight chain alkylene, or alkylene which is substituted by C_M alkyl and n represents 0 to 1 , and an optical isomer thereof or a pharmaceutically acceptable acid addition salt thereof.

11. The method of claim 1 , wherein said cancer is selected from the group consisting of lung cancer, brain cancer, breast cancer, colon cancer, prostate cancer, ovarian cancer, cervical cancer, leukemia, lymphoma, neuroblastoma, skin cancer and pancreatic cancer.

12. In a method of treating cancer in a subject with imatinib or an analog thereof or a pharmaceutically acceptable salt thereof, the improvement comprising: administering said subject a Rho kinase inhibitor in an amount effective to enhance the antineoplastic activity or reduce the neurotoxicity of said imatinib or an analog thereof or a pharmaceutically acceptable salt thereof.

13. The method of claim 12, wherein said Rho kinase inhibitor is Y27632 or an analog thereof.

14. The method of claim 12, wherein said cancer is selected from the group consisting of lung cancer, brain cancer, breast cancer, colon cancer, prostate cancer, ovarian cancer, cervical cancer, leukemia, lymphoma, neuroblastoma, skin cancer and pancreatic cancer.

15. A method of screening for compounds useful for inhibiting neuronal impairment caused by administering an antineoplastic compound comprising: contacting a neuronal cell with a test compound, in the presence of an Abl family tyrosine kinase inhibitor, wherein said contacting step is carried out in vitro; and then determining whether said test compound affects dendrogenesis influenced by Abl family tyrosine kinase inhibition; a positive effect on said dendrogenesis influenced by said Abl family tyrosine kinase inhibition indicates that said test compound is useful for inhibiting neuronal impairment caused by administering an antineoplastic compound.

16. The method of claim 15, wherein said Abl family tyrosine kinase inhibitors inhibit Abl family tyrosine kinases selected from the group consisting of Abl (Abl), v-Abl, BCR-Abl, Arg (Abl-related gene), and Drosophila Abl (D-Abl).

17. The method of claim 15, wherein said Abl family tyrosine kinase inhibitor is imatinib.

18. The method claim 17, wherein said imatinib inhibits Abl family tyrosine kinases, platelet derived growth factor (PDGF) receptor tyrosine kinase or Kit family tyrosine kinases.