TrkA Inhibitor Research Articles

Acquired Resistance to the TRK Inhibitor Entrectinib in Colorectal Cancer.

Entrectinib is a first-in-class pan-TRK kinase inhibitor currently undergoing clinical testing in colorectal cancer and other tumor types. A patient with metastatic colorectal cancer harboring an LMNA-NTRK1 rearrangement displayed a remarkable response to treatment with entrectinib, which was followed by the emergence of resistance. To characterize the molecular bases of the patient's relapse, circulating tumor DNA (ctDNA) was collected longitudinally during treatment, and a tissue biopsy, obtained before entrectinib treatment, was transplanted in mice (xenopatient), which then received the same entrectinib regimen until resistance developed. Genetic profiling of ctDNA and xenopatient samples showed acquisition of two point mutations in the catalytic domain of NTRK1, p.G595R and p.G667C. Biochemical and pharmacologic analysis in multiple preclinical models confirmed that either mutation renders the TRKA kinase insensitive to entrectinib. These findings can be immediately exploited to design next-generation TRKA inhibitors. We provide proof of principle that analyses of xenopatients (avatar) and liquid biopsies allow the identification of drug resistance mechanisms in parallel with clinical treatment of an individual patient. We describe for the first time that p.G595R and p.G667C TRKA mutations drive acquired resistance to entrectinib in colorectal cancers carrying NTRK1 rearrangements.

Abstract PR13: Clinical safety and activity from a phase 1 study of LOXO-101, a selective TRKA/B/C inhibitor, in solid-tumor patients with NTRK gene fusions

Abstract Background: Gene fusions involving the NTRK1, 2 and 3 genes result in constitutively-active TRKA, -B, and -C kinases that are prevalent in a wide array of tumor types, including lung adenocarcinoma, thyroid, head and neck cancer, sarcoma, and other tumors. LOXO-101 is an orally bioavailable, potent, ATP-competitive, selective inhibitor of TRKA, TRKB, and TRKC being studied in a Phase 1 dose escalation trial. In pre-clinical in vivo xenograft mouse models bearing NTRK-fusions LOXO-101 demonstrated potent tumor growth inhibition and regression. Here, we report updated PK and safety data on 24 patients (pts). Methods: In this on-going open-label, multicenter, 3+3 dose escalation Phase I study of LOXO-101, 24 pts with solid tumors refractory to standard therapy, normal hematopoietic and major organ function have been enrolled. LOXO-101 is administered orally as a single dose, followed by QD or BID doses for continuous 28-day cycles. Response is measured by RECIST Criteria, version 1.1. Serum is collected for pharmacokinetic analysis on Cycle 1 Day 1 and Day 8. Safety information is collected on all patients and the definition of dose-limiting toxicity applies to adverse events regardless of relationship to investigational product. Nude mice were injected with CUTO3.29 (MPRIP-NTRK1, lung adenocarcinoma) or MO-91 (ETV6-NTRK3, acute myeloid leukemia) cell lines and then administered LOXO-101 orally at 60 or 200mg/kg daily for 2 weeks. Results: As of September 3, 2015, 24 pts have been treated at each of the first five dose levels (50mg QD, 100mg QD, 200mg QD, 100mg BID, and 150mg BID). LOXO-101 has been well tolerated; the MTD has not been reached and the most common adverse events are Grade 1 and 2 fatigue (42%), dizziness (29%) and anemia (21%). Two pts have had grade 3 AEs leading to dose cohort expansions: elevated AST, grade 3 (Dose Level 150mg BID) and delirium, grade 3 (Dose Level 100mg BID). Each case was subsequently deemed unrelated to study drug by the treating investigator, and the pts remained on study at reduced doses without recurrence. PK analysis showed maximum plasma concentrations of LOXO-101 were reached 30-60 minutes following dosing. The unbound drug levels of LOXO-101 appear sufficient for approximately 98% inhibition of TRKA/B/C at peak concentrations at all dose levels. All three pts with NTRK-fusions enrolled have achieved a PR: an undifferentiated sarcoma with an LMNA-NTRK1 fusion (59% decrease; 7 cycles+), a c-kit-negative GI Stromal Tumor (GIST) with an ETV6-NTRK3 fusion (30% decrease; 2 cycles+), and a mammary analogue secretory carcinoma of the salivary glands with an ETV6-NTRK3 fusion (64% decrease; 2 cycles+). These patients were treated at either 100 or 150 mg BID. These data are supported by in vivo tumor regression in xenograft mouse models of NTRK1 and NTRK3 fusion bearing tumor cells of different histologies in the presence of clinically achievable concentrations of LOXO-101. Conclusions: LOXO-101 has been well tolerated and has sufficient systemic exposure for robust tumor responses in 3 of 3 (100%) of NTRK-fusion pts enrolled on study. These data further validate this molecular target as an oncogenic driver across diverse tumor histologies and support the exploration of TRK inhibition in cancer pts with NTRK-fusions. Citation Format: David S. Hong, Marcia S. Brose, Robert C. Doebele, Alice T. Shaw, Afshin Dowlati, Todd M. Bauer, Anna F. Farago, Adriana Estrada-Bernal, Anh T. Lee, Michael C. Cox, Nisha Nanda, Jennifer A. Low, Howard A. Burris, III. Clinical safety and activity from a phase 1 study of LOXO-101, a selective TRKA/B/C inhibitor, in solid-tumor patients with NTRK gene fusions. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr PR13.

Evaluation of WO2015042088 A1 - a novel urea-based scaffold for TrkA inhibition

ABSTRACTTropomyosin receptor kinases (TrkA/B/C) are involved in the development and maintenance of the nervous system. TrkA is a target for chronic pain treatment due to the central position of the nerve growth factor (NGF)/TrkA pathway in nociception. Clinical evidence points toward mutated oncogenic Trk fusion proteins retaining intact kinase domains as relevant targets for cancer treatment. Merck pursues Trk inhibitors for inflammatory and neuropathic pain treatment and has previously reported type I and II selective pan-Trk inhibitors. This is the fifth filing by Merck disclosing urea-based Trk inhibitor series. This application claims nonsymmetric 1-(9H-fluoren-9-yl)urea and 1-(9H-xanthen-9-yl)urea derivatives containing a wide range of 5- and 6-membered bi- or tri-heterocyclic fragments as TrkA inhibitors for the treatment of Trk-related conditions. The exemplified compounds display IC50 values ranging from 27 to 4800 nM against TrkA. The TrkA inhibitors claimed confirm the emergence of nonsymmetric ureas lacking the hinge-binding motif as a favored Trk inhibitor structure. The compounds exemplified will likely be structurally optimized in the future. Despite the lack of selectivity profiling, the progression of the Trk inhibitor scaffold exploration by Merck also suggests that the compounds disclosed in this patent likely constitute non–adenosine triphosphate (ATP) competitive type III pan-Trk inhibitors.

Cross-talk between lysophosphatidic acid receptor 1 and tropomyosin receptor kinase A promotes lung epithelial cell migration

Lysophosphatidic acid (LPA) is a bioactive lysophospholipid, which plays a crucial role in the regulation of cell proliferation, migration, and differentiation. LPA exerts its biological effects mainly through binding to cell-surface LPA receptors (LPA1-6), which belong to the G protein-coupled receptor (GPCR) family. Recent studies suggest that cross-talk between receptor tyrosine kinases (RTKs) and GPCRs modulates GPCRs-mediated signaling. Tropomyosin receptor kinase A (TrkA) is a RTK, which mediates nerve growth factor (NGF)-induced biological functions including cell migration in neuronal and non-neuronal cells. Here, we show LPA1 transactivation of TrkA in murine lung epithelial cells (MLE12). LPA induced tyrosine phosphorylation of TrkA in both time- and dose-dependent manners. Down-regulation of LPA1 by siRNA transfection attenuated LPA-induced phosphorylation of TrkA, suggesting a cross-talk between LPA1 and TrkA. To investigate the molecular regulation of the cross-talk, we focused on the interaction between LPA1 and TrkA. We found that LPA induced interaction between LPA1 and TrkA. The LPA1/TrkA complex was localized on the plasma membrane and in the cytoplasm. The C-terminus of LPA1 was identified as the binding site for TrkA. Inhibition of TrkA attenuated LPA-induced phosphorylation of TrkA and LPA1 internalization, as well as lung epithelial cell migration. These studies provide a molecular mechanism for the transactivation of TrkA by LPA, and suggest that the cross-talk between LPA1 and TrkA regulates LPA-induced receptor internalization and lung epithelial cell migration.

The neuroprotection of cannabidiol against MPP+-induced toxicity in PC12 cells involves trkA receptors, upregulation of axonal and synaptic proteins, neuritogenesis, and might be relevant to Parkinson's disease

Cannabidiol (CBD) is a non-psychoactive constituent of Cannabis sativa with potential to treat neurodegenerative diseases. Its neuroprotection has been mainly associated with anti-inflammatory and antioxidant events; however, other mechanisms might be involved. We investigated the involvement of neuritogenesis, NGF receptors (trkA), NGF, and neuronal proteins in the mechanism of neuroprotection of CBD against MPP+ toxicity in PC12 cells. CBD increased cell viability, differentiation, and the expression of axonal (GAP-43) and synaptic (synaptophysin and synapsin I) proteins. Its neuritogenic effect was not dependent or additive to NGF, but it was inhibited by K252a (trkA inhibitor). CBD did not increase the expression of NGF, but protected against its decrease induced by MPP+, probably by an indirect mechanism. We also evaluated the neuritogenesis in SH-SY5Y cells, which do not express trkA receptors. CBD did not induce neuritogenesis in this cellular model, which supports the involvement of trkA receptors. This is the first study to report the involvement of neuronal proteins and trkA in the neuroprotection of CBD. Our findings suggest that CBD has a neurorestorative potential independent of NGF that might contribute to its neuroprotection against MPP+, a neurotoxin relevant to Parkinson's disease.

Molecular Mechanism of Switching of TrkA/p75(NTR) Signaling in Monocrotophos Induced Neurotoxicity.

We demonstrate the role of molecular switching of TrkA/p75NTR signaling cascade in organophosphate pesticide-Monocrotophos (MCP) induced neurotoxicity in stem cell derived cholinergic neurons and in rat brain. Our in-silico studies reveal that MCP followed the similar pattern of binding as staurosporine and AG-879 (known inhibitors of TrkA) with TrkA protein (PDB ID: 4AOJ) at the ATP binding sites. This binding of MCP to TrkA led to the conformational change in this protein and triggers the cell death cascades. The in-silico findings are validated by observing the down regulated levels of phosphorylated TrkA and its downstream molecules viz., pERK1/2, pAkt and pCREB in MCP-exposed cells. We observe that these MCP induced alterations in pTrkA and downstream signaling molecules are found to be associated with apoptosis and injury to neurons. The down-regulation of TrkA could be linked to increased p75NTR. The in-vitro studies could be correlated in the rat model. The switching of TrkA/p75NTR signaling plays a central role in MCP-induced neural injury in rBNSCs and behavioral changes in exposed rats. Our studies significantly advance the understanding of the switching of TrkA/p75NTR that may pave the way for the application of TrkA inducer/p75NTR inhibitor for potential therapeutic intervention in various neurodegenerative disorders.

NGF/TrkA Signaling as a Therapeutic Target for Pain.

Nerve growth factor (NGF) was first discovered approximately 60years ago by Rita Levi-Montalcini as a protein that induces the growth of nerves. It is now known that NGF is also associated with Alzheimer's disease and intractable pain, and hence, it, along with its high-affinity receptor, tropomyosin receptor kinase (Trk) A, is considered to be 1 of the new targets for therapies being developed to treat these diseases. Anti-NGF antibody and TrkA inhibitors are known drugs that suppress NGF/TrkA signaling, and many drugs of these classes have been developed thus far. Interestingly, local anesthetics also possess TrkA inhibitory effects. This manuscript describes the development of an analgesic that suppresses NGF/TrkA signaling, which is anticipated to be 1 of the new methods to treat intractable pain.

Blocking the tropomyosin receptor kinase A (TrkA) receptor inhibits pain behaviour in two rat models of osteoarthritis

ObjectivesTropomyosin receptor kinase A (TrkA) mediates nociceptor sensitisation by nerve growth factor (NGF), but it is unknown whether selective TrkA inhibition will be an effective strategy for treating osteoarthritis (OA)...

Abstract 4529: Pharmacokinetics (PK) of LOXO-101 during the first-in-human Phase I study in patients with advanced solid tumors: Interim update

Abstract Background LOXO-101 is the first highly selective and potent small-molecule inhibitor of TRKA, TRKB, and TRKC to be dosed clinically. It has approximately 100-fold selectivity over other kinase and non-kinase targets. We report here the interim results of the first-in-man Phase I study which evaluates the safety, maximum-tolerated dose (MTD), and PK of LOXO-101 in patients with advanced solid tumors. Methods This is an ongoing open-label, multicenter, 3+3 dose escalation Phase I study of LOXO-101. Patients with solid tumors refractory to standard therapy, with normal hematopoietic and major organ function were eligible for study. LOXO-101 was administered orally as a single dose, followed by QD or BID doses for continuous 28-day cycles. Receptor occupancy was determined with an Emax model based on unbound LOXO-101 plasma concentrations. Results To date, data are available for 8 patients including patients treated at each of the first two dose levels, 50 mg QD and 100 mg QD. LOXO-101 has been well tolerated; the MTD has not been reached and no adverse events have been reported more than once other than fatigue (two patients; related but not serious) and syncope (two patients; unrelated and serious). Maximum plasma concentrations of LOXO-101 were reached 30-60 minutes following dosing and exposure increased in approximate proportion with dose. Mean values of Cl/F, V/F, and half-life were 0.5 L/h/kg, 2 L/kg, and 3.5 h, respectively, and were independent of dose. Exposure was similar following the single dose and on Day 1 and Day 8 of repeated dosing in all subjects. The unbound drug levels of LOXO-101 appear sufficient for approximately 98% inhibition of TRKA/B/C at peak concentrations with once-daily dosing of 50 or 100 mg. Conclusions LOXO-101 has been well tolerated and has sufficient systemic exposure for robust inhibition of the TRKA, TRKB, and TRKC kinases without inhibition of other kinases. With dose escalation continuing, additional data and follow up will be provided at time of presentation. Citation Format: Howard A. Burris, Alice T. Shaw, Todd M. Bauer, Anna F. Farago, Robert C. Doebele, Steven Smith, Nisha Nanda, Scott Cruickshank, Jennifer A. Low, Marcia S. Brose. Pharmacokinetics (PK) of LOXO-101 during the first-in-human Phase I study in patients with advanced solid tumors: Interim update. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4529. doi:10.1158/1538-7445.AM2015-4529

Targeting trka inhibition in Alzheimer's disease therapy

Targeting trka inhibition in Alzheimer's disease therapy

Cross-talk between androgen receptor/filamin A and TrkA regulates neurite outgrowth in PC12 cells.

Steroids and growth factors control neuronal development through their receptors under physiological and pathological conditions. We show that PC12 cells harbor endogenous androgen receptor (AR), whose inhibition or silencing strongly interferes with neuritogenesis stimulated by the nonaromatizable synthetic androgen R1881 or NGF. This implies a role for AR not only in androgen signaling, but also in NGF signaling. In turn, a pharmacological TrkA inhibitor interferes with NGF- or androgen-induced neuritogenesis. In addition, androgen or NGF triggers AR association with TrkA, TrkA interaction with PI3-K δ, and downstream activation of PI3-K δ and Rac in PC12 cells. Once associated with AR, filamin A (FlnA) contributes to androgen or NGF neuritogenesis, likely through its interaction with signaling effectors, such as Rac. This study thus identifies a previously unrecognized reciprocal cross-talk between AR and TrkA, which is controlled by β1 integrin. The contribution of FlnA/AR complex and PI3-K δ to neuronal differentiation by androgens and NGF is also novel. This is the first description of AR function in PC12 cells.

Nerve Growth Factor Improves Survival and Function of Transplanted Islets Via TrkA-mediated β Cell Proliferation and Revascularization.

Nerve growth factor (NGF), which plays important roles in promoting growth and differentiation of nerve cells, has recently been reported as a regulator in pancreatic β cells in terms of insulin releasing function. In this study, we examined whether NGF stimulation would promote islet graft survival and function in islet transplantation. We found that supplementation of cultured islets with NGF improved the viability of islet cells and induced the production of insulin, vascular endothelial growth factor, and cellular proliferative markers. Because a specific inhibitor of TrkA, K252a, blocked all these effects, we propose that the TrkA receptor is the mediator of NGF stimulation. After transplantation to the kidney subcapsule and liver of syngenic diabetic mice, a higher rate of normoglycemic achievement, increased serum insulin, and improved glucose tolerance were observed in the mice transplanted with NGF-pretreated islet grafts. Histological analysis revealed higher expression of insulin and vascular endothelial growth factor, an increase in proliferative β cells, and revascularization in NGF-pretreated islet grafts without activation of any inflammatory cells. The NGF treatment can therefore serve as a new and promising therapeutic tool for improving islet graft viability and function in islet transplantation.

Efficacy of a novel, locally delivered TrkA inhibitor in preclinical models of OA and joint pain

Purpose: OA and associated joint pain is a major cause of disability, and there is a high unmet need for effective and safe analgesic therapies. Substantial pain alleviation has been reported for OA patients treated with intravenous (IV) infusions of antibodies which block nerve growth factor (NGF), however an increased drug-related risk for accelerated OA progression was observed in some patients. As an alternate strategy for targeting this pathway, we identified a novel small molecule compound, GZ389988, as an inhibitor of TrkA, the high-affinity receptor for NGF. GZ389988 was formulated for intraarticular (IA) delivery, and its effects on local knee pain were tested in rats using both a monosodium iodoacetate (MIA) induced model of OA, as well as an arthritis flare model mediated by streptococcal peptidoglycan polysaccharide (PGPS). Methods: Identification of TrkA inhibitor GZ389988: Drug candidate screening was conducted to select inhibitors of Trk family tyrosine kinases. Low solubility compounds were evaluated using a cell-based TrkA assay, and further screened against kinase and safety/liability panels. Acute cytotoxicity was assessed by neutral red assay for a variety of cell types, including primary human chondrocytes and synoviocytes (Articular Engineering). In vivo biocompatibility was tested by IA injection of GZ389988 into rat knee joints. Preclinical rat OA model: Unilateral knee OA was initiated by IA injection of MIA. One week later, diseased (ipsilateral) joints were treated with a single IA injection of GZ389988 or placebo/vehicle, and differences in hind-limb weight distribution were measured weekly for 4 weeks by incapacitance testing. In some cases, GZ389988 was administered to the contralateral knee instead, to test for the occurrence of systemic compound distribution affecting the ipsilateral joint. Preclinical rat arthritis flare model (Bolder BioPATH): Unilateral primary knee arthritis was induced by IA injection of PGPS. Two weeks later, animals received a single IA injection of GZ389988 or placebo/vehicle into the diseased (ipsilateral) joint. Disease reactivation flares were triggered by IV administration of PGPS at weeks 1 and 3 following drug (or placebo) treatment. Gait analysis was conducted by obtaining inked pawprint impressions from animals which were allowed to ambulate freely along a papered walkway. Gait scoring and quantification was performed using 4 distinct pawprint pairs per animal, collected daily for 4 days following each reactivation. Results: GZ389988 was identified as a potent inhibitor of TrkA (similar IC50 values observed for TrkA, TrkB, TrkC and cFMS/CSF1R), with a high level of selectivity against a wide panel of other kinases. In addition, GZ389988 has very low aqueous solubility, thus facilitating its formulation for local administration. No cytotoxicity was observed in cell-based assays using criteria of LD50/IC50 ratio >100. In vivo biocompatibility studies with GZ389988 showed neither a treatment effect on body weight or clinical appearance, nor any significant histopathologic changes in joint tissues. In the rat MIA model, a single ipsilateral dose of GZ389988 provided a significant reduction in local joint pain (weight-bearing imbalance) for 4 weeks. Treatment of the contralateral joint with GZ389988 had no effect on ipsilateral joint pain (Fig. 1), illustrating the absence of a substantial systemic effect following IA administration. GZ389988 was also efficacious in the PGPS arthritis model, demonstrating a significant improvement of gait analysis scores following painful disease reactivation flares (Fig. 2). Conclusions: Elevated levels of NGF are associated with tissue injury, and have been observed in a number of painful conditions including OA. In addition, TrkA is expressed by OA synovial fibroblasts and is upregulated in response to NGF. In OA pain trials, NGF-blocking Abs have demonstrated significant efficacy with high patient responder rates, however such studies have been subject to clinical hold by the FDA due to the occurrence of joint-related adverse events. We have targeted the NGF pain signaling pathway using a locally-effective inhibitor of TrkA. In addition to the efficacy data presented here, pharmacokinetic studies and safety pharmacology and preclinical toxicology data support the development of GZ389988 as a novel therapeutic approach for the treatment of OA and joint pain.Figure 2. Efficacy of GZ389988 in rat PGPS model of joint pain. Disease was inititated by a single IA injection of PGPS into the knee joint (ipsilateral) three weeks prior to the first reactivation. A single dose of GZ389988 was administered by IA injection into the knee one week prior to the first reactivation. Disease reactivation (painful flare) was induccd on study days 0 and 14 by IV administration of PGPS. Gait analysis was performed based on pawprint areas, and scored as follows: 1= mild limping/pain; 2=moderate limping/pain; 3=marked limping/pain; 4=severe hopping/pain. Data are presented as mean + SEM.View Large Image Figure ViewerDownload Hi-res image Download (PPT)

The association of obesity with intervertebral disc degeneration, disc herniation and spinal stenosis: A MRI study of 1,684 patients

The association of obesity with intervertebral disc degeneration, disc herniation and spinal stenosis: A MRI study of 1,684 patients

Th2 cytokines enhance TrkA expression, upregulate proliferation, and downregulate differentiation of keratinocytes

BackgroundNerve growth factor (NGF), a neurotrophin that plays a critical role in developmental neurobiology, is released by proliferating keratinocytes and induces proliferation. ObjectiveThe aim of this study was to investigate the role of tyrosine kinase receptor A (TrkA), a high-affinity receptor of NGF, in human keratinocytes. MethodsExpression of TrkA and NGF in skin diseases was investigated by immunohistochemistry. Expression of TrkA in cells was examined by Western blotting and RT-PCR. Cell proliferation was assessed by BrdU assay. ResultsWe first determined the expression of TrkA and NGF in skin samples from patients with atopic dermatitis, prurigo nodularis, psoriasis vulgaris, and seborrheic keratosis. TrkA was only expressed in proliferating basal cells, and its expression was enhanced in atopic dermatitis samples. NGF expression was enhanced in atopic dermatitis and prurigo nodularis samples and in some samples from seborrheic keratosis patients. Investigation of the role of TrkA in vitro using normal human epidermal keratinocytes (NHEK) revealed that TrkA was significantly enhanced by the T helper type 2 (Th2) cytokines interleukin (IL)-4 and IL-13 but not by other inflammatory cytokines, such as IL-1β, tumor necrosis factor α, interferon γ, or epidermal growth factor. On the other hand, expression of NGF was not altered by Th2 cytokines. Notably, inhibition of TrkA significantly reversed the effects of IL-4 on proliferation and differentiation. Furthermore, overexpression of TrkA enhanced proliferation of NHEK. These results indicate that IL-4-induced TrkA expression in keratinocytes modulates proliferation and differentiation of these cells. ConclusionIncreased TrkA expression in keratinocytes in atopic dermatitis may contribute to the observed epidermal hyperproliferation in these patients.

Urinary bladder inflammation induces changes in urothelial nerve growth factor and TRPV1 channels.

The urinary bladder urothelium expresses various receptors and in response to chemical and mechanical stimuli releases mediators, thereby modulating bladder sensory pathways. Transient receptor potential vanilloid 1 (TRPV1) ion channels and nerve growth factor (NGF) in those cells are implicated in this modulatory effect and play a role in sensitizing pain-related afferent pathways during inflammation. In this study, we investigated the interaction between NGF and TRPV1 channels in urothelial cells. Urothelial cells from female Sprague-Dawley rat bladders were cultured to quantify membrane expression of TRPV1 channels and capsaicin-induced ATP release in the presence of NGF alone or with TrKA or PI3K inhibitors. Pain scores from rats with cyclophosphamide (CYP)-induced bladder inflammation were assessed after treatment with a TrkA antagonist. Bladders (from control and CYP rats) were collected and analysed for NGF content and TRPV1 channel expression. Cultured cells responded to NGF with increased TRPV1 channel expression in the cell membrane and increased release of ATP. Both responses were blocked by either a TrkA antagonist or a PI3K inhibitor. Treatment in vivo with the TrkA antagonist alleviated pain symptoms and reduced CYP-induced NGF overexpression in the mucosa. Furthermore, in urothelial cells from animals with bladder inflammation, expression of TRPV1 channels in the membrane was significantly increased. During bladder inflammation, increased production of NGF in urothelial cells induced increased expression and activity of TRPV1 channels in the cell membrane. This effect was primarily mediated by the PI3K pathway.

Nerve growth factor protects the ischemic heart via attenuation of the endoplasmic reticulum stress induced apoptosis by activation of phosphatidylinositol 3-kinase.

Background: Increased expression of nerve growth factor (NGF) has been found in the myocardium suffered from ischemia and reperfusion (I/R). The pro-survival activity of NGF on ischemic heart has been supposed to be mediated by phosphatidylinositol 3-kinase/protein kinase B (PI3K/Akt) signaling pathway. Endoplasmic reticulum (ER) stress, which is activated initially as a defensive response to eliminate the accumulated unfolded proteins, has shown a critical involvement in the ischemia induced myocardial apoptosis. This study was aimed to investigate whether NGF induced heart protection against I/R injury includes a mechanism of attenuation of ER stress-induced myocardial apoptosis by activation of PI3K/Akt pathway.Methods: Isolated adult rat hearts were perfused with a Langendörff perfusion system. Hearts in the Sham group were subjected to 225 min of continuous Krebs-Henseleit buffer (KHB) perfusion without ischemia. Hearts in I/R group were perfused with KHB for a 75-min of equilibration period followed by 30 min of global ischemia and 120 min of KHB reperfusion. Hearts in the NGF group accepted 45 min of euilibration perfusion and 30 min of NGF pretreatment (with a final concentration of 100 ng/ml in the KHB) before 30 min of global ischemia and 120 min of reperfusion. Hearts in K252a and LY294002 groups were pretreated with either a TrkA inhibitor, K252a or a phosphatidyl inositol 3-kinase inhibitor, LY294002 for 30 min before NGF (100 ng/ml) administration. Cardiac hemodynamics were measured from the beginning of the perfusion. Cardiac enzymes and cardiac troponin I (cTnI) were assayed before ischemia and at the end of reperfusion. Myocardial apoptosis rate was measured by TUNEL staining, and expression of glucose-related protein 78 (GRP78), CCAAT/enhancer-binding protein homologous protein (CHOP), caspase-12, total- and phospho-(Ser473)-Akt were assessed by Western blot analyses.Results: NGF pretreatment significantly improved the recovery of post-ischemia cardiac hemodynamics. Reduced creatine kinase-MB (CK-MB), lactate dehydrogenase (LDH) activity and cTnI levels, as well as decreased myocardial apoptosis ratio were observed in the NGF group. The improvement of NGF on recovery of cardiac function and alleviation of myocardial injury were completely abolished by K252a or LY294002. GRP78, caspase-12 and CHOP were highly expressed in ischemic myocardium, while NGF significantly inhibited the overexpression of these proteins which were involved in ER stress-induced myocardial apoptosis. NGF pretreatment also induced phosphorylation of Akt. When the activation of PI3K/Akt pathway is blocked by LY294002, the NGF induced suppression of the apoptosis-related proteins expression was reversed.Conclusions: NGF pretreatment may protect the ischemic heart via inhibition of the ER stress-induced apoptosis; this pro-survival effect is mediated by PI3K/Akt pathway.

Trka, a Novel Binding Partner of NPM-ALK Oncogenic Tyrosine Kinase, Facilitates the Survival of T-Cell Anaplastic Large-Cell Lymphoma

Anaplastic lymphoma kinase-expressing (ALK+) T-cell anaplastic large-cell lymphoma (ALCL) is an aggressive type of cancer that frequently occurs in children and adolescents. Approximately 85% of the ALK+ ALCL patients harbor the translocation t(2;5)(p23;q35), which generates the chimeric oncogene NPM-ALK. The expression of ALK is primarily identified in neural tissues at early stages of human development, which suggests that ALK contributes to neural tissue development probably through interactions with neurotrophic factors.Nerve growth factor (NGF) is the first characterized neurotrophic factor. In addition to its canonical roles in neural tissue development, NGF has gained attention as a promoter of survival of cancer cells. Indeed, deregulation of NGF signaling has been attributed to cell proliferation, invasion, and metastasis. It is believed that NGF’s non-neural functions are essentially mediated through two cognate receptors: TrkA and p75NTR. The expression and role of NGF/TrkA signaling in NPM-ALK+ T-cell lymphoma have not been previously studied. Because of the biological similarities between ALK and NGF/TrkA signaling, we hypothesized that blockade of NGF/TrkA signaling may suppress NPM-ALK+ T-cell lymphoma cell survival, which can have important therapeutic implications.Western blotting and immunohistochemical staining showed that TrkA and NGF proteins are highly expressed in 5 NPM-ALK+ T-cell lymphoma cell lines compared with normal human T lymphocytes. In addition, immunohistochemical staining demonstrated the expression of TrkA in 85% (11/13) of ALK+ T-cell lymphoma patient tumors. Immunoprecipitation showed physical association between TrkA and NPM-ALK. Specific downregulation of NPM-ALK using ALK siRNA was associated with a marked decrease in phosphorylated TrkA, suggesting functional interactions between the two proteins. Thereafter, we used a selective TrkA Inhibitor (catalogue number: 648450; Calbiochem, Billerica, MA) and performed in vitro assays to elucidate the contribution of TrkA signaling on the lymphoma cells. The TrkA Inhibitor decreased efficiently the phosphorylation and kinase activity of TrkA in NPM-ALK+ T-cell lymphoma cells. These effects were associated with decreased NPM-ALK+ T-cell lymphoma cellular proliferation. The TrkA Inhibitor also induced apoptosis and abrogated anchorage-independent colony formation of these cells. Moreover, the TrkA Inhibitor caused downregulation of pNPM-ALK, pIGF-IR, pSTAT3, and pAKT. Caspase-3, BCL-2 and BCL-XL were also reduced, supporting apoptosis occurrence. To rule out nonspecific effects that might have been caused by the TrkA Inhibitor, TrkA siRNA was used in some experiments. TrkA siRNA, but not scrambled siRNA, decreased the viability and proliferation of NPM-ALK+ T-cell lymphoma cells. In addition, TrkA siRNA caused biochemical effects similar to those observed with the TrkA Inhibitor. An ELISA assay demonstrated the presence of NGF in cell culture supernatant of serum-deprived NPM-ALK+ lymphoma cells. Importantly, NGF enhanced the viability and proliferation of NPM-ALK+ T-cell lymphoma cells, and TrkA blocking antibody abrogated these effects. These results suggest the presence of an autocrine NGF/TrkA loop that supports the survival of NPM-ALK+ T-cell lymphoma. We also examined the effects of TrkA Inhibitor in SCID-beige mice with NPM-ALK+ T-cell lymphoma xenografts implanted subcutaneously using SR-786 cells. The tumors were established after 8 days, and mice were randomly treated with vehicle or the TrkA Inhibitor for 21 days (10 mg/kg b.i.d; s.c.). Tumor volumes were remarkably smaller in the TrkA Inhibitor-treated mice compared with vehicle-treated mice.Our data provide novel evidence that TrkA contributes to the survival of NPM-ALK+ T-cell lymphoma, and implicate that targeting TrkA may represent an effective approach to suppress this lymphoma. DisclosuresNo relevant conflicts of interest to declare.

Hepatic nerve growth factor induced by iron overload triggers defenestration in liver sinusoidal endothelial cells

The fenestrations of liver sinusoidal endothelial cells (LSECs) play important roles in the exchange of macromolecules, solutes, and fluid between blood and surrounding liver tissues in response to hepatotoxic drugs, toxins, and oxidative stress. As excess iron is a hepatotoxin, LSECs may be affected by excess iron. In this study, we found a novel link between LSEC defenestration and hepatic nerve growth factor (NGF) in iron-overloaded mice. By Western blotting, NGF was highly expressed, whereas VEGF and HGF were not, and hepatic NGF mRNA levels were increased according to digital PCR. Immunohistochemically, NGF staining was localized in hepatocytes, while TrkA, an NGF receptor, was localized in LSECs. Scanning electron microscopy revealed LSEC defenestration in mice overloaded with iron as well as mice treated with recombinant NGF. Treatment with conditioned medium from iron-overloaded primary hepatocytes reduced primary LSEC fenestrations, while treatment with an anti-NGF neutralizing antibody or TrkA inhibitor, K252a, reversed this effect. However, iron-loaded medium itself did not reduce fenestration. In conclusion, iron accumulation induces NGF expression in hepatocytes, which in turn leads to LSEC defenestration via TrkA. This novel link between iron and NGF may aid our understanding of the development of chronic liver disease.

ET-30 * A PHASE I TRIAL OF AZD7451, A TROPOMYOSIN-RECEPTOR KINASE (TRK) INHIBITOR, FOR ADULTS WITH RECURRENT GLIOBLASTOMA MULTIFORME (GBM)

BACKGROUND: We have shown that inhibition of TrkA both genetically and pharmacologically results in profound inhibition of glioma stem cell migration in vitro and in vivo. AZD7451 is the first in class of highly potent and selective Trk inhibitors and therefore may have significant anti-glioma cell invasive properties. METHODS: We conducted an open label phase I study of AZD 7451 mesylate, with a standard 3 + 3 dose escalation scheme, to determine the MTD and describe pharmacokinetics. The drug was administered as an oral solution (10 mg/ml) at 12 mg, 24 mg, 40 mg, and 60 mg daily. DL3 and DL4 were administered with divided twice daily dosing. Plasma concentrations of AZD7451 were measured with ultra-performance liquid chromatography and tandem mass spectrometry using samples obtained at baseline and various points through the first 4 week cycle of therapy. RESULTS: 14 patients were enrolled. There were no radiographic responses to therapy, and no patients achieved disease control beyond cycle 2. There were no DLTs. Unexpected treatment related toxicities included grade 1-2 thrombocytopenia and anemia. No neurological adverse events related to therapy were observed. All patients were evaluable for C1D1 pharmacokinetics. The majority of samples were below the limit of quantitation (LLOQ) of 0.5 ng/mL by ∼ 4 h post-dose. AZD7451 T1/2 ranged from 20-50 min with once daily or BID dosing. No statistically significant increases in CMAX, AUCINF, T1/2, or volume of distribution were observed as dose increased. CONCLUSIONS: Based on the interim PK analysis for AZD7451 indicating poor oral bioavailability, short <60 min plasma T1/2 half-life, and rapid clearance kinetics, PK exposures are insufficient in humans to inhibit Trk at target plasma concentrations necessary for efficacy in preclinical models. The absence of DLT's or early efficacy signals in GBM patients is also consistent with poor AZD7451 exposures in humans.