Naquotinib exerts antitumor activity in activated B-cell-like diffuse large B-cell lymphoma.

Diffuse large B-cell lymphoma (DLBCL), the most common type of B-cell non-Hodgkin lymphoma (NHL), is categorized into two major subtypes, activated B-cell-like (ABC) and germinal center B-cell-like (GCB). The ABC subtype is associated with worse prognosis than the GCB subtype using currently available therapies such as combination treatment with rituximab plus standard cytotoxic chemotherapy. The B-cell receptor (BCR) pathway is activated in ABC DLBCL, suggesting that inhibition of this pathway could provide an alternative strategy for treatment. Naquotinib is an irreversible tyrosine kinase inhibitor (TKI) originally designed to target the epidermal growth factor receptor (EGFR). As sequence alignment analysis indicates that irreversible EGFR-TKIs also inhibit Bruton's tyrosine kinase (BTK), here, we characterized the inhibitory effects of naquotinib against BTK in comparison to ibrutinib, acalabrutinib, tirabrutinib and spebrutinib. Naquotinib inhibited BTK kinase activity with similar potency to that for EGFR activating mutations. In vivo, naquotinib induced tumor regression and suppressed tumor recurrence in TMD8 and OCI-Ly10, ABC DLBCL cell line xenograft models, at a lower dose than the clinically relevant dose. Compared to other BTK inhibitors, naquotinib showed faster onset and comparable inhibition of BTK following incubation with cell lines for 3 and 20 h. In addition, naquotinib showed longer continuous inhibition of BTK following removal of the compound, lasting for at least 26 h after removal. Pharmacokinetics studies in the TMD8 xenograft model showed higher concentration and slower elimination of naquotinib in tumors than other BTK inhibitors. These data suggest that naquotinib may have therapeutic potential in ABC DLBCL patients.

Mutant-Selective Irreversible EGFR Inhibitor, Naquotinib, Inhibits Tumor Growth in NSCLC Models with EGFR-Activating Mutations, T790M Mutation, and AXL Overexpression.

First- and second-generation EGFR tyrosine kinase inhibitors (TKI) are effective clinical therapies for patients with non-small cell lung cancer (NSCLC) harboring EGFR-activating mutations. However, almost all patients develop resistance to these drugs. The EGFR T790M mutation of EGFR is the most predominant mechanism for resistance. In addition, activation of AXL signaling is one of the suggested alternative bypassing pathways for resistance to EGFR-TKIs. Here, we report that naquotinib, a pyrazine carboxamide-based EGFR-TKI, inhibited EGFR with activating mutations, as well as T790M resistance mutation while sparing wild-type (WT) EGFR. In in vivo murine xenograft models using cell lines and a patient-derived xenograft model, naquotinib induced tumor regression of NSCLC with EGFR-activating mutations with or without T790M resistance mutation, whereas it did not significantly inhibit WT EGFR signaling in skin. Furthermore, naquotinib suppressed tumor recurrence during the treatment period of 90 days. In addition, unlike erlotinib and osimertinib, naquotinib inhibited the phosphorylation of AXL and showed antitumor activity against PC-9 cells overexpressing AXL in vitro and in vivo Our findings suggest that naquotinib has therapeutic potential in patients with NSCLC with EGFR-activating mutations, T790M resistance mutation, and AXL overexpression.

Synthesis and structure-activity relationships of pyrazine-2-carboxamide derivatives as novel echinoderm microtubule-associated protein-like 4 (EML4)-anaplastic lymphoma kinase (ALK) inhibitors.

Echinoderm microtubule-associated protein-like 4 (EML4)-anaplastic lymphoma kinase (ALK) is a valid therapeutic target for the treatment of EML4-ALK-positive non-small cell lung cancer (NSCLC). We discovered 12c as a novel and potent EML4-ALK inhibitor through structural optimization of 5a. In mice xenografted with 3T3 cells expressing EML4-ALK, oral administration of 12c demonstrated potent antitumor activity. This article describes the synthesis and biological evaluation of pyrazine-2-carboxamide derivatives along with studies of their structure-activity relationship (SAR) using computational modeling.

Discovery of N-{2-Methoxy-4-[4-(4-methylpiperazin-1-yl)piperidin-1-yl]phenyl}-N'-[2-(propane-2-sulfonyl)phenyl]-1,3,5-triazine-2,4-diamine (ASP3026), a Potent and Selective Anaplastic Lymphoma Kinase (ALK) Inhibitor.

Anaplastic lymphoma kinase (ALK) is a validated therapeutic target for treating echinoderm microtubule-associated protein-like 4 (EML4)-ALK positive non-small cell lung cancer (NSCLC). We synthesized a series of 1,3,5-triazine derivatives and identified ASP3026 (14a) as a potent and selective ALK inhibitor. In mice xenografted with NCI-H2228 cells expressing EML4-ALK, once-daily oral administration of 14a demonstrated dose-dependent antitumor activity. Here, syntheses and structure-activity relationship (SAR) studies of 1,3,5-triazine derivatives are described.

Radiosynthesis, biodistribution and imaging of [11C]YM155, a novel survivin suppressant, in a human prostate tumor-xenograft mouse model.

INTRODUCTION: Sepantronium bromide (YM155) is an antitumor drug in development and is a first-in-class chemical entity, which is a survivin suppressant. We developed a radiosynthesis of [(11)C]YM155 to non-invasively evaluate its tissue and tumor distribution in mice bearing human prostate tumor xenografts. METHODS: Methods utilizing [(11)C]acetyl chloride and [(11)C]methyl triflate, both accessible with automated radiosynthesis boxes, were evaluated. The O-methylation of ethanolamine-alkolate with [(11)C]methyl triflate proved to be the key development toward a rapid and efficient process. The whole-body distribution of [(11)C]YM155 in PC-3 xenografted mice was examined using a planar positron imaging system (PPIS). RESULTS: Sufficient quantities of radiopharmaceutical grade [(11)C]YM155 were produced for our PET imaging and distribution studies. The decay corrected (EOB) radiochemical yield was 16-22%, within a synthesis time of 47 min. The radiochemical purity was higher than 99%, and the specific activity was 29-60 GBq/µmol (EOS). High uptake levels of radioactivity (%ID/g, mean±SE) were observed in tumor (0.0613±0.0056), kidneys (0.0513±0.0092), liver (0.0368±0.0043) and cecum (0.0623±0.0070). The highest tumor uptake was observed at an early time point (from 10 min after) following injection. Tumor-to-blood and tumor-to-muscle uptake ratios of [(11)C]YM155, at 40 min after injection, were 26.5 (±2.9) and 25.6 (±3.6), respectively. CONCLUSION: A rapid method for producing a radiopharmaceutical grade [(11)C]YM155 was developed. An in vivo distribution study using PPIS showed high uptake of [(11)C]YM155 in tumor tissue. Our methodology may facilitate the evaluation and prediction of response to YM155, when given as an anti-cancer agent.

Brain adenosine A2A receptor occupancy by a novel A1/A2A receptor antagonist, ASP5854, in rhesus monkeys: relationship to anticataleptic effect.

UNLABELLED: The purpose of the present study was to measure adenosine A(2A) receptor (A(2A)R) occupancy in the brain by a novel adenosine A(1)/A(2A) antagonist, 5-[5-amino-3-(4fluorophenyl)pyrazin-2-yl]-1-isopropylpyridine-2(1H)-one (ASP5854), and to determine the degree of receptor occupancy necessary to inhibit haloperidol-induced catalepsy in rhesus monkeys. METHODS: A(2A)R occupancy by ASP5854 (0.001-0.1 mg/kg) was examined in the striatum using an A(2A)R-specific radiotracer, (11)C-SCH442416, and PET in conscious rhesus monkeys. A(2A)R occupancy was monitored after a single intravenous administration of ASP5854 in 3 animals, and a dynamic PET scan was performed at 1, 4, and 8 h after an intravenous bolus injection of the tracer for approximately 740 MBq. Catalepsy was induced by haloperidol (0.03 mg/kg, intramuscularly) and examined for incidence and duration. RESULTS: ASP5854 dose-dependently increased A(2A)R occupancy in the striatum and showed long-lasting occupancy even after the reduction of plasma concentration. Haloperidol induced severe catalepsy at 40 min after intramuscular injection. The incidence and duration of cataleptic posture were dose-dependently reduced by ASP5854 at 1 h after oral administration, and the minimum ED(50) value was 0.1 mg/kg. Administration of a dose of 0.1 mg/kg yielded a plasma concentration of 97 +/- 16.3 ng/mL, which corresponded to 85%-90% of A(2A)R occupancy. CONCLUSION: These results showed that ASP5854 antagonized A(2A)R in the striatum, and the dissociation from A(2A)R was relatively slow. In addition, more than 85% A(2A)R occupancy by ASP5854 resulted in an inhibition of haloperidol-induced catalepsy. Thus, such a pharmacodynamic study directly demonstrates both the kinetics of a drug in the brain and the relationship between dose-dependent receptor occupancy and plasma level.

Synergistic effects of adenosine A2A antagonist and L-DOPA on rotational behaviors in 6-hydroxydopamine-induced hemi-Parkinsonian mouse model.

In this study, we examined the combination effects of L-DOPA and adenosine receptor antagonists on rotational behaviors in a hemi-Parkinsonian mouse model induced by unilateral 6-hydroxydopamine (6-OHDA) injection. The adenosine A(2A) antagonist SCH-58261, but not the A(1)-receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine or A(2B)-receptor antagonist alloxazine, synergistically potentiated the L-DOPA-induced rotational behaviors in the 6-OHDA-lesioned mice. In addtion, the 6-OHDA-induced lesions of the dopaminergic system did not affect the in vivo binding of an adenosine A(2A)-receptor tracer [(11)C]SCH-442416 in the striatatum. These findings suggest that adenosine A(2A) antagonists are extremely useful for pharmacotherapy of L-DOPA in Parkinson's disease patients.

Neural circuits containing pallidotegmental GABAergic neurons are involved in the prepulse inhibition of the startle reflex in mice.

BACKGROUND: Prepulse inhibition (PPI) of the startle response is a measure of the inhibitory function and time-linked information processing by which a weak sensory stimulus (the prepulse) inhibits the startle response caused by a sudden intense stimulus. We attempted to clarify the neuronal circuits underlying the control of PPI of the startle reflex in mice. METHODS: c-Fos immunohistochemistry was used to detect neurons activated by startle pulse and/or prepulse trials. Behavioural pharmacology and tracing studies were also conducted. RESULTS: The lateral globus pallidus (LGP) was activated by prepulses. Activation of the caudal pontine reticular nucleus (PnC) evoked by the startle pulses was inhibited under PPI conditions. Double-immunostaining revealed that c-Fos-positive cells in the LGP following prepulse trials were GABAergic neurons. Bilateral microinjections of lidocaine into the LGP resulted in an impairment of PPI. Fluoro-gold infusion into the PnC and the pedunculopontine tegmental nucleus (PPTg) retrogradely labeled neurons in the PPTg and LGP, respectively. Microinjections of phaclofen into the PPTg significantly impaired PPI. CONCLUSIONS: These results suggest that GABAergic neurons in the LGP which project to the PPTg play a crucial role through the activation of GABAB receptors in the regulation of PPI of the startle reflex in mice.

Involvement of hippocampal extracellular signal-regulated kinase 1/2 in spatial working memory in rats.

The role of the phosphorylation of hippocampal extracellular signal-regulated kinase 1/2 in spatial working memory in rats was assessed with a delayed spatial win-shift task in a radial arm maze. The task consisted of two phases, a training phase and a test phase, separated by a delay. Phosphorylated extracellular signal-regulated kinase 1/2 levels were significantly and transiently increased in the hippocampus by 60 min, and then returned to the control levels 120 min after the training phase. Bilateral microinjections of the PD98059, an inhibitor of the extracellular signal-regulated kinase 1/2 kinase MEK, into the hippocampus impaired performance in the test phase of the delayed spatial win-shift task at 5-min delay. These results suggest that extracellular signal-regulated kinase 1/2 activation in the hippocampus plays a crucial role in spatial working memory.

Quantitation of cardiac sympathetic innervation in rabbits using 11C-hydroxyephedrine PET: relation to 123I-MIBG uptake.

PURPOSE: Although (11)C-hydroxyephedrine ((11)C-HED) PET is used to map cardiac sympathetic innervation, no studies have shown the feasibility of quantitation of (11)C-HED PET in small- to medium-sized animals. Furthermore, its relation to (123)I-MIBG uptake, the most widely used sympathetic nervous tracer, is unknown. The aims of this study were to establish in vivo sympathetic nerve imaging in rabbits using (11)C-HED PET, and to compare the retention of (11)C-HED with that of (123)I-MIBG. METHODS: Twelve rabbits were assigned to three groups; control (n=4), chemical denervation by 6-hydroxydopamine (6-OHDA) (n=4) and reserpine treated to inhibit vesicular uptake (n=4). After simultaneous injection of (11)C-HED and (123)I-MIBG, all animals underwent dynamic (11)C-HED PET for 40 min with arterial blood sampling. The (11)C-HED retention fraction and normalised (11)C-HED activity measured by tissue sampling were compared with those measured by PET. RESULTS: Both the (11)C-HED retention fraction and the normalised (11)C-HED activity measured by PET correlated closely with those measured by tissue sampling (R=0.96027, p<0.001 and R=0.97282, p<0.001, respectively). Inhibition study by 6-OHDA resulted in a significant reduction in retention (90%) for both (11)C-HED and (123)I-MIBG. Reserpine pretreatment reduced (11)C-HED retention by 50%, but did not reduce (123)I-MIBG retention at 40 min after injection. CONCLUSION: Non-invasive quantitation of cardiac sympathetic innervation using (11)C-HED PET is feasible and gives reliable estimates of cardiac sympathetic innervation in rabbits. Additionally, although both (11)C-HED and (123)I-MIBG are specific for sympathetic neurons, (11)C-HED may be more specific for intravesicular uptake than (123)I-MIBG in some situations, such as that seen in reserpine pretreatment.

Synthesis and evaluation of [11C]FR194921 as a nonxanthine-type PET tracer for adenosine A1 receptors in the brain.

This report describes the synthesis of [11C]2-(1-methyl-4-piperidinyl)-6-(2-phenylpyrazolo[1,5-a]pyridin-3-yl)-3(2H)-pyridazinone ([11C]FR194921), a highly selective, nonxanthine-type adenosine A(1) receptor antagonist, used in brain imaging in rats and conscious monkeys as a potential novel PET tracer. [11C]FR194921 was successfully synthesized in 19 min after [11C]CH3I formation. The radiochemical yield was 38+/-3%; and radioactivity was 4.1+/-0.4 GBq, calculated from end of synthesis; radiochemical purity was higher than 99%; and the specific radioactivity was 25.0+/-8.1 GBq micromol(-1) (n=5). In a rat experiment, the distribution of [11C]FR194921 was higher in the hippocampus, striatum and cerebellum regions. This accumulation was significantly decreased by approximately 50% by pretreatment with 8-cyclopentyl-1,3-dipropylxanthine (DPCPX), an adenosine A1 receptor antagonist, which indicated specific binding of the radioligand to adenosine A1 receptors. In conscious monkey PET experiments, [11C]FR194921 accumulated in several regions of the brain, especially in the occipital cortex, thalamus and striatum. These results suggest that [11C]FR194921 can be used as an agent for imaging adenosine A1 receptors in vivo by positron emission tomography (PET).