Acquisition of cocaine reinforcement using fixed-ratio and concurrent choice schedules in socially housed female and male monkeys.

RATIONALE: Previous studies in socially housed monkeys examining acquisition of cocaine self-administration under fixed-ratio (FR) schedules of reinforcement found that subordinate males and dominant females were more vulnerable than their counterparts. OBJECTIVES: The present studies extended these findings in two ways: (1) to replicate the earlier study, in which female monkeys were studied after a relatively short period of social housing (~ 3 months) using cocaine-naïve female monkeys (n = 9; 4 dominant and 5 subordinate) living in well-established social groups (~ 18 months); and (2) in male monkeys (n = 3/social rank), we studied cocaine acquisition under a concurrent schedule, with an alternative, non-drug reinforcer available. RESULTS: In contrast to earlier findings, subordinate female monkeys acquired cocaine reinforcement (i.e., > saline reinforcement) at significantly lower cocaine doses compared with dominant monkeys. In the socially housed males, no dominant monkey acquired a cocaine preference (i.e., > 80% cocaine choice) over food, while two of three subordinate monkeys acquired cocaine reinforcement. In monkeys that did not acquire, the conditions were changed to an FR schedule with only cocaine available and after acquisition, returned to the concurrent schedule. In all monkeys, high doses of cocaine were chosen over food reinforcement. CONCLUSIONS: The behavioral data in females suggests that duration of social enrichment and stress can differentially impact vulnerability to cocaine reinforcement. The findings in socially housed male monkeys, using concurrent food vs. cocaine choice schedules of reinforcement, confirmed earlier social-rank differences using an FR schedule and showed that vulnerability could be modified by exposure to cocaine.

A comparison of the reinforcing strength of cocaethylene and cocaine in monkeys responding under progressive-ratio and concurrent choice schedules of reinforcement.

BACKGROUND: Individuals who use cocaine have high rates of co-morbid alcohol use and when ethanol and cocaine are administered concurrently, the metabolite cocaethylene is formed. Cocaethylene is equipotent to cocaine in blocking dopamine reuptake and substitutes for cocaine in drug discrimination studies. However, no previous work has directly compared the reinforcing strength of cocaine to cocaethylene. METHODS: In Experiment 1, three individually-housed adult male rhesus macaques self-administer cocaine under a progressive-ratio (PR) schedule of reinforcement, during daily 4-hr sessions. Under this schedule, the primary dependent variable is the number of injections received, or the break point (BP). Saline, cocaine (0.001-0.3mg/kg/injection) and cocaethylene (0.0003-0.1mg/kg/injection) dose-response curves were determined. In Experiment 2, two female cynomolgus and one rhesus macaque responded under a concurrent schedule of drug (cocaine or cocaethylene) vs. 1.0-g banana-flavored food pellets, during daily 1-hr sessions. RESULTS: Both cocaine and cocaethylene functioned as reinforcers under the PR and concurrent choice schedules of reinforcement. Under the PR schedule, peak BPs were not significantly different, nor were ED50 values on the ascending limb, suggesting that cocaethylene has equal reinforcing strength and potency to cocaine. Under the concurrent drug-food choice procedure, cocaethylene was also equally potent to cocaine. CONCLUSIONS: Under two schedules of reinforcement designed to assess reinforcing strength, cocaethylene and cocaine were equipotent and of equal reinforcing strength. Because cocaethylene has a longer duration of action, it is important for studies designed to evaluate treatments for cocaine use to also consider the effects of these interventions on cocaethylene.

Delay discounting as a behavioral phenotype associated with social rank in female and male cynomolgus monkeys: Correlation with kappa opioid receptor availability.

Cocaine use disorder (CUD) is a significant problem worldwide, with no FDA-approved treatments. Epidemiological data indicate that only about 17 % of people that use cocaine will meet DSM criteria for CUD. Thus, the identification of biomarkers predictive of eventual cocaine use may be of great value. Two potentially useful predictors of CUD are social hierarchies in nonhuman primates and delay discounting. Both social rank and preference for a smaller, immediate reinforcer relative to a larger, delayed reinforcer have been predictive of CUD. Therefore, we wanted to determine if there was also a relationship between these two predictors of CUD. In the present study, monkeys cocaine-naive responded under a concurrent schedule of 1- vs. 3-food pellets and delivery of the 3-pellet option was delayed. The primary dependent variable was the indifference point (IP), which is the delay that results in 50 % choice for both options. In the initial determination of IP, there were no differences based on sex or social rank of the monkeys. When the delays were redetermined after ~25 baseline sessions (range 5-128 sessions), dominant females and subordinate males showed the largest increases in IP scores from the first determination to the second. Because 13 of these monkeys had prior PET scans of the kappa opioid receptor (KOR), we examined the relationship between KOR availability and IP values and found that the change in IP scores from the first to the second determination significantly negatively predicted average KOR availability in most brain regions. Future studies will examine acquisition to cocaine self-administration in these same monkeys, to determine if IP values are predictive of vulnerability to cocaine reinforcement.

PET imaging of kappa opioid receptors and receptor expression quantified in neuron-derived extracellular vesicles in socially housed female and male cynomolgus macaques.

Recent positron emission tomography (PET) studies of kappa opioid receptors (KOR) in humans reported significant relationships between KOR availability and social status, as well as cocaine choice. In monkey models, social status influences physiology, receptor pharmacology and behavior; these variables have been associated vulnerability to cocaine abuse. The present study utilized PET imaging to examine KOR availability in socially housed, cocaine-naïve female and male monkeys, and peripheral measures of KORs with neuron-derived extracellular vesicles (NDE). KOR availability was assessed in dominant and subordinate female and male cynomolgus macaques (N = 4/rank/sex), using PET imaging with the KOR selective agonist [11C]EKAP. In addition, NDE from the plasma of socially housed monkeys (N = 13/sex; N = 6-7/rank) were isolated by immunocapture method and analyzed for OPRK1 protein expression by ELISA. We found significant interactions between sex and social rank in KOR availability across 12 of 15 brain regions. This was driven by female data, in which KOR availability was significantly higher in subordinate monkeys compared with dominant monkeys; the opposite relationship was observed among males, but not statistically significant. No sex or rank differences were observed for NDE OPRK1 concentrations. In summary, the relationship between brain KOR availability and social rank was different in female and male monkeys. This was particularly true in female monkeys. We hypothesize that lower [11C]EKAP binding potentials were due to higher concentrations of circulating dynorphin, which is consistent with greater vulnerability in dominant compared with subordinate females. These findings suggest that the KOR is an important target for understanding the neurobiology associated with vulnerability to abused drugs and sex differences, and detectable in peripheral circulation.

Selective TAAR1 agonists induce conditioned taste aversion.

RATIONALE: Trace amine-associated receptor 1 (TAAR1) is the best-studied receptor of trace amines, a group of biogenic amines expressed at a relatively low level in the mammalian brain. Growing evidence suggests that TAAR1 plays a critical role in various neuropsychiatric disorders. Given that selective TAAR1 agonists were shown to produce pro-cognition and antipsychotic-like effects as well as to suppress drug use and relapse, they have been proposed to be novel treatments for mental disorders such as schizophrenia and addiction. However, the aversive effects of selective TAAR1 agonists remain largely unknown. OBJECTIVES: Here, we evaluated whether the selective TAAR1 full agonist RO5166017 and partial agonist RO5263397 could induce conditioned taste aversion (CTA). RESULTS: We found that RO5166017 and RO5263397 produced significant aversions to both saccharin and NaCl taste novelty. Furthermore, RO5166017 produced CTA to saccharin in TAAR1 heterozygous knockout (taar1±) and wild-type rats but not in TAAR1 homozygous knockout rats (taar1-/-), suggesting that TAAR1 was sufficient for the taste aversive stimulus property of RO5166017. CONCLUSIONS: Taken together, our data indicate that selective TAAR1 agonists could produce strong CTA. Our study urges careful evaluations of the aversive effects of TAAR1 agonists before translating them to clinical use for the treatment of mental disorders.

TAAR1 regulates drug-induced reinstatement of cocaine-seeking via negatively modulating CaMKIIα activity in the NAc.

Relapse remains a major challenge to the treatment of cocaine addiction. Recent studies suggested that the trace amine-associated receptor 1 (TAAR1) could be a promising target to treat cocaine addiction and relapse; however, the underlying mechanism remains unclear. Here, we aimed to investigate the neural mechanism underlying the role of TAAR1 in the drug priming-induced reinstatement of cocaine-seeking behavior in rats, an animal model of cocaine relapse. We focused on the shell subregion of nucleus accumbens (NAc), a key brain region of the brain reward system. We found that activation of TAAR1 by systemic and intra-NAc shell administration of the selective TAAR1 agonist RO5166017 attenuated drug-induced reinstatement of cocaine-seeking and prevented drug priming-induced CaMKIIα activity in the NAc shell. Activation of TAAR1 dampened the CaMKIIα/GluR1 signaling pathway in the NAc shell and reduced AMPAR-EPSCs on the NAc slice. Microinjection of the selective TAAR1 antagonist EPPTB into the NAc shell enhanced drug-induced reinstatement as well as potentiated CaMKIIα activity in the NAc shell. Furthermore, viral-mediated expression of CaMKIIα in the NAc shell prevented the behavioral effects of TAAR1 activation. Taken together, our findings indicate that TAAR1 regulates drug-induced reinstatement of cocaine-seeking by negatively regulating CaMKIIα activity in the NAc. Our findings elucidate a novel mechanism of TAAR1 in regulating drug-induced reinstatement of cocaine-seeking and further suggests that TAAR1 is a promising target for the treatment of cocaine relapse.

Activation of trace amine-associated receptor 1 attenuates nicotine withdrawal-related effects.

Nicotine addiction is a leading avoidable brain disorder globally. Although nicotine induces a modest reinforcing effect, which is important for the initial drug use, the transition from nicotine use to nicotine addiction involves the mechanisms responsible for the negative consequences of drug abstinence. Recent study suggested that trace amine-associated receptor 1 (TAAR1) is a promising pharmacological target for the modulation of positive reinforcing effects of nicotine. However, whether TAAR1 plays a part in the negative reinforcement of nicotine withdrawal remains to be determined. Here, using a long-access (LA) self-administration model, we investigated whether LA rats show increased nicotine intake and withdrawal symptoms in comparison with saline and ShA rats and then tested the effect of TAAR1 partial agonist RO5263397 on nicotine withdrawal effects. We found that rats from long-access group showed significant abstinence-induced anxiety-like behaviour, mechanic hypersensitivity, increased number of precipitated withdrawal signs and higher motivation for the drug, while rats from short-access did not differ from saline group. TAAR1 partial agonist RO5263397 significantly reduced the physical and motivational withdrawal effects of nicotine in LA rats, as reflected by increased time spent on the open arm in the elevated plus maze (EPM) test, normalized paw withdrawal threshold, decreased withdrawal signs and motivation to self-administer nicotine. This study indicates that activation of TAAR1 attenuates the negative-reinforcing effects of nicotine withdrawal and further suggests TAAR1 as a promising target to treat nicotine addiction.

The selective TAAR1 partial agonist RO5263397 promoted novelty recognition memory in mice.

RATIONALE: Trace amine-associated receptor 1 (TAAR1) is a G protein-coupled receptor that has a particular role in regulating dopaminergic, serotonergic, and glutamatergic transmission. TAAR1 agonists have shown pro-cognitive activities. However, it remains largely unknown of the effects of TAAR1 agonists on memory performance. OBJECTIVES: Here, by using the mice novel object recognition (NOR) test, we examined the effects of the selective TAAR1 partial agonist RO5263397 on recognition memory. RESULTS: We found that RO5263397 significantly enhanced the retrieval of short-term memory (STM; 20 min after training) both in male and female mice. RO5263397 promoted the retrieval of STM in the wild-type (WT) littermates but not TAAR1-KO mice, indicating that the effects of RO5263397 were dependent on TAAR1. Interestingly, compared to their WT litters, TAAR1-KO mice showed similar levels of STM, suggesting that genetic deletion of taar1 gene did not affect the STM retrieval. Furthermore, RO5263397 also promoted the retrieval of long-term NOR memory (24 h after training). CONCLUSIONS: These results indicate that TAAR1 activation promotes NOR memory retrieval. Consistent with previous studies, our finding further suggests that TAAR1 agonists have pro-cognitive properties.

Activation of trace amine-associated receptor 1 selectively attenuates the reinforcing effects of morphine.

BACKGROUND AND PURPOSE: Trace amine-associated TA1 receptors play critical roles in regulating dopamine transmission. Previous studies showed that pharmacologically or genetically manipulating the activity of TA1 receptors modulates addiction-like behaviours associated with psychostimulants. However, little is known about whether TA1 receptor modulation would regulate the behavioural effects of opioids. EXPERIMENTAL APPROACH: Effects of the selective TA1 receptor partial agonist RO5263397 on the addiction-related and antinociceptive effects of morphine were systematically assessed in male rats and mice. KEY RESULTS: RO5263397 attenuated the expression of morphine-induced behavioural sensitization in wildtype but not TA1 receptor knockout mice. RO5263397 shifted the dose-effect curve of morphine self-administration downward and reduced the breakpoint in a progressive ratio schedule of reinforcement but did not affect food self-administration in rats. RO5263397 decreased the cue- and drug-induced reinstatement of morphine-seeking behaviour in rats. RO5263397 alone did not trigger reinstatement of morphine-seeking behaviour or change locomotor activity in rats with a history of morphine self-administration. However, RO5263397 did not affect the expression of morphine-induced conditioned place preference in mice or rats. RO5263397 did not affect naltrexone-precipitated jumping behaviour or naltrexone-induced conditioned place aversion in morphine-dependent mice. Furthermore, RO5263397 did not affect the analgesic effects of morphine in an acute nociception model in mice and a chronic pain model in rats. CONCLUSION AND IMPLICATIONS: These results indicated that TA1 receptor activation selectively attenuated the reinforcing, but not withdrawal or antinociceptive effects of morphine, suggesting that selective TA1 receptor agonists might be useful to combat opioid addiction, while sparing the analgesic effects.

TAAR1 agonists attenuate extended-access cocaine self-administration and yohimbine-induced reinstatement of cocaine-seeking.

BACKGROUND AND PURPOSE: The trace amine-associated receptor 1 (TAAR1) negatively modulates dopamine transmission. Our previous studies demonstrated that TAAR1 agonists attenuated cue- and drug-induced cocaine-seeking and increased the elasticity of the cocaine demand curve, in the short-access cocaine self-administration model. Compulsive use of cocaine, which is an essential criterion of cocaine use disorder, can be induced by extended access to cocaine self-administration. EXPERIMENTAL APPROACH: To characterize the role of TAAR1 in compulsive cocaine use, we evaluated the effects of activation of TAAR1 on cocaine intake, cocaine binge and cue-induced cocaine-seeking using the extended-access cocaine self-administration model in adult male Sprague-Dawley rats. We also investigated the role of TAAR1 in stress-triggered cocaine relapse by using the α2 -adrenoceptor antagonist yohimbine-induced reinstatement of cocaine-seeking. KEY RESULTS: The selective TAAR1 partial agonist RO5263397 attenuated cocaine intake and did not develop tolerance during the 10-day extended-access cocaine self-administration. RO5263397 reduced a 12-h binge intake of cocaine after forced abstinence. RO5263397 also decreased cue-induced cocaine-seeking after prolonged abstinence from extended-access cocaine self-administration. Furthermore, RO5263397 and the selective TAAR1 full agonist RO5166017 reduced yohimbine-induced reinstatement of cocaine-seeking behaviour. CONCLUSION AND IMPLICATIONS: Activation of TAAR1 attenuated extended-access cocaine self-administration and stress-induced cocaine reinstatement. These results suggest that TAAR1 agonists are promising pharmacological interventions to treat cocaine use disorder and relapse.

The Claustrum-Prefrontal Cortex Pathway Regulates Impulsive-Like Behavior.

The claustrum connects with a broad range of cortical areas including the prefrontal cortex (PFC). However, the function of the claustrum (CLA) and its neural projections remains largely unknown. Here, we elucidated the role of the neural projections from the CLA to the PFC in regulating impulsivity in male rats. We first identified the CLA-PFC pathway by retrograde tracer and virus expression. By using immunofluorescent staining of the c-Fos-positive neurons, we showed that chemogenetic activation and inhibition of the CLA-PFC pathway reduced and increased overall activity of the PFC, respectively. In the 5-choice serial reaction time task (5-CSRTT), we found that chemogenetic activation and inhibition of the CLA-PFC pathway increased and reduced the impulsive-like behavior (i.e., premature responses), respectively. Furthermore, chemogenetic inhibition of the CLA-PFC pathway prevented methamphetamine-induced impulsivity, without affecting methamphetamine-induced hyperactivity. In contrast to the role of CLA-PFC pathway in selectively regulating impulsivity, activation of the claustrum disrupted attention in the 5-CSRTT. These results indicate that the CLA-PFC pathway is essential for impulsivity. This study may shed light on the understanding of impulsivity-related disorders such as drug addiction.SIGNIFICANCE STATEMENT The claustrum is one of the most mysterious brain regions. Although extensive anatomical studies demonstrated that the claustrum connects with many cortical areas, the function of the neural projections between the claustrum and cortical areas remain largely unknown. Here, we showed that the neural projections from the claustrum to the prefrontal cortex regulates impulsivity by using the designer drugs (DREADDs)-based chemogenetic tools. Interestingly, the claustrum-prefrontal cortex pathway also regulates methamphetamine-induced impulsivity, suggesting a critical role of this neural pathway in regulating impulsivity-related disorders such as drug addiction. Our results provided preclinical evidence that the claustrum-prefrontal cortex regulates impulsivity. The claustrum-prefrontal cortex pathway may be a novel target for the treatment of impulsivity-related brain disorders.

Role of trace amine-associated receptor 1 in nicotine's behavioral and neurochemical effects.

Nicotine addiction and abuse remains a global health issue. To date, the fundamental neurobiological mechanism of nicotine addiction remains incompletely understood. Trace amine-associated receptor 1 (TAAR1) is thought to directly modulate dopaminergic system and are thought to be a neural substrate underlying addictive-like behaviors. We aimed to investigate the role of TAAR1 in nicotine addictive-like behaviors. TAAR1 expression after nicotine treatment was evaluated by western blotting. c-Fos immunofluorescence and in vivo fast-scan cyclic voltammetry were used to examine the activation of brain regions and dopamine release, respectively. We then thoroughly and systematically examined the role of TAAR1 in mediating nicotine-induced sensitization, nicotine discrimination, nicotine self-administration, nicotine demand curve, and the reinstatement of nicotine-seeking. Local pharmacological manipulation was conducted to determine the role of TAAR1 in the nucleus accumbens (NAcs) in the reinstatement of nicotine-seeking. We found that the expression of TAAR1 protein was selectively downregulated in the NAc, with no change in either dorsal striatum or prefrontal cortex. TAAR1 activation was sufficient to block nicotine-induced c-Fos expression in the NAc, while also reducing nicotine-induced dopamine release in the NAc. Systemic administration of TAAR1 agonists attenuated the expression and development of nicotine-induced sensitization, nicotine self-administration, the reinstatement of nicotine-seeking, and increased the elasticity of nicotine demand curve, while intra-NAc infusions of a TAAR1 agonist was sufficient to attenuate nicotine reinstatement. Moreover, TAAR1-knockout rats showed augmented cue-induced and drug-induced reinstatement of nicotine-seeking. These results indicated that modulation of TAAR1 activity regulates nicotine addictive-like behaviors and TAAR1 represents a novel target towards the treatment of nicotine addiction.

Methamphetamine-induced impulsivity during chronic methamphetamine treatment in rats: Effects of the TAAR 1 agonist RO5263397.

Impulsivity is an important personality trait associated with several clinical syndromes including drug abuse. While repeated drug exposure is known to increase certain behavioral responses, such as locomotion, to subsequent drug exposure, few studies have examined whether such sensitization develops for impulsive behavior. In the current study we tested the effects of methamphetamine acutely, during the course of, and upon discontinuation of chronic methamphetamine treatment on impulsive behavior in two models, the 5-choice serial reaction time task (5-CSRTT) and the delay-discounting task which measure impulsive action and impulsive choice, respectively. We also examined whether the trace amine-associated receptor 1 (TAAR1) agonist RO5263397 attenuated methamphetamine-induced effects in parallel tests. Acute methamphetamine dose-dependently increased premature responses in the 5-CSRTT and shifted the delay function upward in delay discounting. Up to 40 days of methamphetamine treatment did not significantly alter the dose-effect curve of methamphetamine-induced premature responses, but produced a significant effect in the delay-discounting task. RO5263397 attenuated acute methamphetamine-induced premature responses, but this effect became non-significant over the course of chronic treatment. RO5263397 did not significantly alter the delay-discounting performance. Discontinuation of methamphetamine treatment increased premature responses, which was attenuated by RO5263397, but did not significantly alter the delay discounting function. These results suggest that acute discontinuation from prolonged methamphetamine treatment increases impulsivity, which can be reduced by a TAAR1 agonist.

Pyranonaphthoquinone lactones: a new class of AKT selective kinase inhibitors alkylate a regulatory loop cysteine.

The naturally occurring pyranonaphthoquinone (PNQ) antibiotic lactoquinomycin and related aglycones were found to be selective inhibitors of the serine-threonine kinase AKT. A set of synthetic PNQs were prepared and a minimum active feature set and preliminary SAR were determined. PNQ lactones inhibit the proliferation of human tumor cell lines containing constitutively activated AKT and show expected effects on cellular biomarkers. Biochemical data are presented supporting a proposed bioreductive alkylation mechanism of action.

Genetic variation in OAS1 is a risk factor for initial infection with West Nile virus in man.

West Nile virus (WNV) is a re-emerging pathogen that can cause fatal encephalitis. In mice, susceptibility to WNV has been reported to result from a single point mutation in oas1b, which encodes 2'-5' oligoadenylate synthetase 1b, a member of the type I interferon-regulated OAS gene family involved in viral RNA degradation. In man, the human ortholog of oas1b appears to be OAS1. The 'A' allele at SNP rs10774671 of OAS1 has previously been shown to alter splicing of OAS1 and to be associated with reduced OAS activity in PBMCs. Here we show that the frequency of this hypofunctional allele is increased in both symptomatic and asymptomatic WNV seroconverters (Caucasians from five US centers; total n = 501; OR = 1.6 [95% CI 1.2-2.0], P = 0.0002 in a recessive genetic model). We then directly tested the effect of this SNP on viral replication in a novel ex vivo model of WNV infection in primary human lymphoid tissue. Virus accumulation varied markedly among donors, and was highest for individuals homozygous for the 'A' allele (P<0.0001). Together, these data identify OAS1 SNP rs10774671 as a host genetic risk factor for initial infection with WNV in humans.

Genetic deficiency of chemokine receptor CCR5 is a strong risk factor for symptomatic West Nile virus infection: a meta-analysis of 4 cohorts in the US epidemic.

West Nile virus (WNV) causes disease in approximately 20% of infected humans. We previously reported that homozygosity for CCR5Delta32, a nonfunctional variant of chemokine receptor CCR5, is markedly increased among symptomatic WNV-seropositive patients from Arizona and Colorado. To confirm this, we analyzed cohorts from California and Illinois. An increase in CCR5-deficient subjects was found in both (for California, odds ratio [OR], 4.2 [95% confidence interval {CI}, 1.5-11.9] [P= .004]; for Illinois, OR, 3.1 [95% CI, 0.9-11.2] [P= .06]). A meta-analysis of all 4 cohorts showed an OR of 4.2 (95% CI, 2.1-8.3 [P= .0001]). Thus, CCR5 deficiency is a strong and consistent risk factor for symptomatic WNV infection in the United States.

Identification of anthranilic acid derivatives as a novel class of allosteric inhibitors of hepatitis C NS5B polymerase.

A series of potent anthranilic acid-based inhibitors of the hepatitis C NS5B polymerase has been identified. The inhibitors bind to a site on NS5B between the thumb and palm regions adjacent to the active site as determined by X-ray crystallography of the enzyme-inhibitor complex. Guided by both molecular modeling and traditional SAR, the enzyme activity of the initial hit was improved by approximately 100-fold, yielding a series of potent and selective NS5B inhibitors with IC50 values as low as 10 nM. These compounds were also inhibitors of the HCV replicon in cultured HUH7 cells.

2-(Quinazolin-4-ylamino)-[1,4]benzoquinones as covalent-binding, irreversible inhibitors of the kinase domain of vascular endothelial growth factor receptor-2.

A series of 2-(quinazolin-4-ylamino)-[1,4] benzoquinone derivatives that function as potent covalent-binding, irreversible inhibitors of the kinase domain of vascular endothelial growth factor receptor-2 (VEGFR-2) has been prepared by ceric ammonium nitrate oxidation of substituted (2,5-dimethoxyphenyl)(6,7-disubstituted-quinazolin-4-yl)amines and by displacement of the chlorine atom of substituted 2-chloro-5-(6,7-disubstituted-quinazolin-4-ylamino)-[1,4]benzoquinones with various amines, anilines, phenols, and alcohols. Enzyme studies were conducted in the absence and presence of glutathione and plasma. Several of the compounds inhibit VEGF-stimulated autophosphorylation in intact cells. Kinetic experiments were performed to study the reactivity of selected inhibitors toward glutathione. Reactivities correlated with LUMO energies calculated as averages of those of individual conformers weighted by the Boltzmann distribution. These results and molecular modeling were used to rationalize the biological observations. The compounds behave as non-ATP-competitive inhibitors. Unequivocal evidence, from mass spectral studies, indicates that these inhibitors form a covalent interaction with Cys-1045. One member of this series displays antitumor activity in an in vivo model.

Optimization of 6,7-disubstituted-4-(arylamino)quinoline-3-carbonitriles as orally active, irreversible inhibitors of human epidermal growth factor receptor-2 kinase activity.

A series of new 6,7-disubstituted-4-(arylamino)quinoline-3-carbonitrile derivatives that function as irreversible inhibitors of human epidermal growth factor receptor-2 (HER-2) and epidermal growth factor receptor (EGFR) kinases have been prepared. These compounds demonstrated enhanced activities for inhibiting HER-2 kinase and the growth of HER-2 positive cells compared to our EGFR kinase inhibitor 86 (EKB-569). Three synthetic routes were used to prepare these compounds. They were prepared mostly by acylation of 6-amino-4-(arylamino)quinoline-3-carbonitriles with unsaturated acid chlorides or by amination of 4-chloro-6-(crotonamido)quinoline-3-carbonitriles with monocyclic or bicyclic anilines. The third route was developed to prepare a key intermediate, 6-acetamido-4-chloroquinoline-3-carbonitrile, that involved a safer cyclization step. We show that attaching a large lipophilic group at the para position of the 4-(arylamino) ring results in improved potency for inhibiting HER-2 kinase. We also show the importance of a basic dialkylamino group at the end of the Michael acceptor for activity, due to intramolecular catalysis of the Michael addition. This, along with improved water solubility, resulted in compounds with enhanced biological properties. We present molecular modeling results consistent with the proposed mechanism of inhibition. Binding studies of one compound, 25o (C-14 radiolabeled), showed that it binds irreversibly to HER-2 protein in BT474 cells. Furthermore, it demonstrated excellent oral activity, especially in HER-2 overexpressing xenografts. Compound 25o (HKI-272) was selected for further studies and is currently in phase I clinical trials for the treatment of cancer.

Antitumor activity of HKI-272, an orally active, irreversible inhibitor of the HER-2 tyrosine kinase.

HER-2 belongs to the ErbB family of receptor tyrosine kinases, which has been implicated in a variety of cancers. Overexpression of HER-2 is seen in 25-30% of breast cancer patients and predicts a poor outcome in patients with primary disease. Trastuzumab (Herceptin), a monoclonal antibody to HER-2, is specifically approved for HER-2-positive breast cancer but is active only in a subset of these tumors. Blocking HER-2 function by a small molecule kinase inhibitor, therefore, represents an attractive alternate strategy to inhibit the growth of HER-2-positive tumors. HKI-272 is a potent inhibitor of HER-2 and is highly active against HER-2-overexpressing human breast cancer cell lines in vitro. It also inhibits the epidermal growth factor receptor (EGFR) kinase and the proliferation of EGFR-dependent cells. HKI-272 reduces HER-2 receptor autophosphorylation in cells at doses consistent with inhibition of cell proliferation and functions as an irreversible binding inhibitor, most likely by targeting a cysteine residue in the ATP-binding pocket of the receptor. In agreement with the predicted effects of HER-2 inactivation, HKI-272 treatment of cells results in inhibition of downstream signal transduction events and cell cycle regulatory pathways. This leads to arrest at the G(1)-S (Gap 1/DNA synthesis)-phase transition of the cell division cycle, ultimately resulting in decreased cell proliferation. In vivo, HKI-272 is active in HER-2- and EGFR-dependent tumor xenograft models when dosed orally on a once daily schedule. On the basis of its favorable preclinical pharmacological profile, HKI-272 has been selected as a candidate for additional development as an antitumor agent in breast and other HER-2-dependent cancers.