Identification of a Potent, Highly Selective, and Brain Penetrant Phosphodiesterase 2A Inhibitor Clinical Candidate.

Computational modeling was used to direct the synthesis of analogs of previously reported phosphodiesterase 2A (PDE2A) inhibitor 1 with an imidazotriazine core to yield compounds of significantly enhanced potency. The analog PF-05180999 (30) was subsequently identified as a preclinical candidate targeting cognitive impairment associated with schizophrenia. Compound 30 demonstrated potent binding to PDE2A in brain tissue, dose responsive mouse brain cGMP increases, and reversal of N-methyl-d-aspartate (NMDA) antagonist-induced (MK-801, ketamine) effects in electrophysiology and working memory models in rats. Preclinical pharmacokinetics revealed unbound brain/unbound plasma levels approaching unity and good oral bioavailability resulting in an average concentration at steady state (Cav,ss) predicted human dose of 30 mg once daily (q.d.). Modeling of a modified release formulation suggested that 25 mg twice daily (b.i.d.) could maintain plasma levels of 30 at or above targeted efficacious plasma levels for 24 h, which became part of the human clinical plan.

Professional Competencies ToolKit: Using Flash Cards to Teach Reflective Practice to Medical Students in Clinical Clerkship.

Introduction: Early clinical experiences can be overwhelming to medical students. The Professional Competencies ToolKit (ProComp ToolKit) gives medical students a framework on which to build these early experiences and reflect on issues related to professionalism as each patient encounter unfolds. Methods: The ProComp ToolKit is a set of 28 flash cards, grouped within six domains of professional competency. Each flash card is a tool for learning, defining a topic in a catchy title and laying out a specific set of skills to be acquired within the clinical setting. Tasks and tips on each flash card guide students through the process of clinical observation, self-observation, and patient interactions. Students meet in small groups with facilitators every 2 weeks throughout the year to discuss these experiences. At the end of the year, students write a narrative based on the flash card that was most meaningful to their professional identity development. Results: We demonstrated how the student narratives that emerged from using the flash cards, exchanged in a small-group setting, led to group problem solving and validation of students' experiences and values. In the narratives, students discussed the origin of negative behaviors and attitudes that can become normalized in patient care while asserting the primacy of patient-centered care and devising self-awareness strategies. Discussion: Our experience using the ProComp ToolKit shows that teaching reflective practice can successfully be integrated into students' clinical experiences. Professionalism skills can be reflected upon such that they become habitual and integral to students' developing professional identities.

The discovery and characterization of the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor potentiator N-{(3S,4S)-4-[4-(5-cyano-2-thienyl)phenoxy]tetrahydrofuran-3-yl}propane-2-sulfonamide (PF-04958242).

A unique tetrahydrofuran ether class of highly potent α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor potentiators has been identified using rational and structure-based drug design. An acyclic lead compound, containing an ether-linked isopropylsulfonamide and biphenyl group, was pharmacologically augmented by converting it to a conformationally constrained tetrahydrofuran to improve key interactions with the human GluA2 ligand-binding domain. Subsequent replacement of the distal phenyl motif with 2-cyanothiophene to enhance its potency, selectivity, and metabolic stability afforded N-{(3S,4S)-4-[4-(5-cyano-2-thienyl)phenoxy]tetrahydrofuran-3-yl}propane-2-sulfonamide (PF-04958242, 3), whose preclinical characterization suggests an adequate therapeutic index, aided by low projected human oral pharmacokinetic variability, for clinical studies exploring its ability to attenuate cognitive deficits in patients with schizophrenia.

Reduction of brain kynurenic acid improves cognitive function.

The elevation of kynurenic acid (KYNA) observed in schizophrenic patients may contribute to core symptoms arising from glutamate hypofunction, including cognitive impairments. Although increased KYNA levels reduce excitatory neurotransmission, KYNA has been proposed to act as an endogenous antagonist at the glycine site of the glutamate NMDA receptor (NMDAR) and as a negative allosteric modulator at the α7 nicotinic acetylcholine receptor. Levels of KYNA are elevated in CSF and the postmortem brain of schizophrenia patients, and these elevated levels of KYNA could contribute to NMDAR hypofunction and the cognitive deficits and negative symptoms associated with this disease. However, the impact of endogenously produced KYNA on brain function and behavior is less well understood due to a paucity of pharmacological tools. To address this issue, we identified PF-04859989, a brain-penetrable inhibitor of kynurenine aminotransferase II (KAT II), the enzyme responsible for most brain KYNA synthesis. In rats, systemic administration of PF-04859989 dose-dependently reduced brain KYNA to as little as 28% of basal levels, and prevented amphetamine- and ketamine-induced disruption of auditory gating and improved performance in a sustained attention task. It also prevented ketamine-induced disruption of performance in a working memory task and a spatial memory task in rodents and nonhuman primates, respectively. Together, these findings support the hypotheses that endogenous KYNA impacts cognitive function and that inhibition of KAT II, and consequent lowering of endogenous brain KYNA levels, improves cognitive performance under conditions considered relevant for schizophrenia.

Inhibition of phosphodiesterase 10A has differential effects on dopamine D1 and D2 receptor modulation of sensorimotor gating.

RATIONALE: Inhibitors of phosphodiesterase 10A (PDE10A), an enzyme highly expressed in medium spiny neurons of the mammalian striatum, enhance activity in direct (dopamine D1 receptor-expressing) and indirect (D2 receptor-expressing striatal output) pathways. The ability of such agents to act to potentiate D1 receptor signaling while inhibiting D2 receptor signaling suggest that PDE10A inhibitors may have a unique antipsychotic-like behavioral profile differentiated from the D2 receptor antagonist-specific antipsychotics currently used in the treatment of schizophrenia. OBJECTIVES: To evaluate the functional consequences of PDE10A inhibitor modulation of D1 and D2 receptor pathway signaling, we compared the effects of a PDE10A inhibitor (TP-10) on D1 and D2 receptor agonist-induced disruptions in prepulse inhibition (PPI), a measure of sensorimotor gating disrupted in patients with schizophrenia. RESULTS: Our results indicate that, in rats: (1) PDE10A inhibition (TP-10, 0.32-10.0 mg/kg) has no effect on PPI disruption resulting from the mixed D1/D2 receptor agonist apomorphine (0.5 mg/kg), confirming previous report; (2) Yet, TP-10 blocked the PPI disruption induced by the D2 receptor agonist quinpirole (0.5 mg/kg); and attenuated apomorphine-induced disruptions in PPI in the presence of the D1 receptor antagonist SCH23390 (0.005 mg/kg). CONCLUSIONS: These findings indicate that TP-10 cannot block dopamine agonist-induced deficits in PPI in the presence of D1 activation and suggest that the effect of PDE10A inhibition on D1 signaling may be counterproductive in some models of antipsychotic activity. These findings, and the contribution of TP-10 effects in the direct pathway on sensorimotor gating in particular, may have implications for the potential antipsychotic efficacy of PDE10A inhibitors.

Pharmacological disruption of mouse social approach behavior: relevance to negative symptoms of schizophrenia.

Social withdrawal is one of several negative symptoms of schizophrenia, all of which are poorly treated by current therapies. One challenge in developing agents with efficacy against negative symptoms is the lack of suitable preclinical models. The social approach test was used as the basis for developing an assay to test emerging therapies for negative symptoms. NMDA antagonists and dopamine agonists have been used extensively to produce or disrupt behaviors thought to be rodent correlates of positive and cognitive symptoms of schizophrenia. The aim of these studies was to determine whether sociability of mice in the 3-chamber social approach test could be disrupted and whether this paradigm could have utility in predicting efficacy against negative symptoms. The criteria for such a model were: a lack of response to antipsychotics and attenuation by agents such as the glycine agonist, d-cycloserine, which has been shown to possess clinical efficacy against negative symptoms. Administration of the NMDA antagonists MK-801, PCP, or ketamine did not disrupt sociability. In contrast, Grin1 hypomorph mice displayed a social deficit which was not reversed by atypical antipsychotics or d-serine. d-Amphetamine disrupted sociability without stimulating locomotor activity and its effect was not reversed by antipsychotics. The GABAA inverse agonist, FG-7142, reduced sociability and this was reversed by the GABAA antagonist, flumazenil and dcycloserine, but not by clozapine, or the GABAA benzodiazepine anxiolytic, alprazolam. Based on our criteria, the GABAA model warrants further evaluation to confirm that this paradigm has utility as a preclinical model for predicting efficacy against negative symptoms of schizophrenia.

Phosphodiesterase 9A regulates central cGMP and modulates responses to cholinergic and monoaminergic perturbation in vivo.

Cyclic nucleotides are critical regulators of synaptic plasticity and participate in requisite signaling cascades implicated across multiple neurotransmitter systems. Phosphodiesterase 9A (PDE9A) is a high-affinity, cGMP-specific enzyme widely expressed in the rodent central nervous system. In the current study, we observed neuronal staining with antibodies raised against PDE9A protein in human cortex, cerebellum, and subiculum. We have also developed several potent, selective, and brain-penetrant PDE9A inhibitors and used them to probe the function of PDE9A in vivo. Administration of these compounds to animals led to dose-dependent accumulation of cGMP in brain tissue and cerebrospinal fluid, producing a range of biological effects that implied functional significance for PDE9A-regulated cGMP in dopaminergic, cholinergic, and serotonergic neurotransmission and were consistent with the widespread distribution of PDE9A. In vivo effects of PDE9A inhibition included reversal of the respective disruptions of working memory by ketamine, episodic and spatial memory by scopolamine, and auditory gating by amphetamine, as well as potentiation of risperidone-induced improvements in sensorimotor gating and reversal of the stereotypic scratching response to the hallucinogenic 5-hydroxytryptamine 2A agonist mescaline. The results suggested a role for PDE9A in the regulation of monoaminergic circuitry associated with sensory processing and memory. Thus, PDE9A activity regulates neuronal cGMP signaling downstream of multiple neurotransmitter systems, and inhibition of PDE9A may provide therapeutic benefits in psychiatric and neurodegenerative diseases promoted by the dysfunction of these diverse neurotransmitter systems.

Modulation of NMDA receptor function by inhibition of D-amino acid oxidase in rodent brain.

Observations that N-Methyl-D-Aspartate (NMDA) antagonists produce symptoms in humans that are similar to those seen in schizophrenia have led to the current hypothesis that schizophrenia might result from NMDA receptor hypofunction. Inhibition of D-amino acid oxidase (DAAO), the enzyme responsible for degradation of D-serine, should lead to increased levels of this co-agonist at the NMDA receptor, and thereby provide a therapeutic approach to schizophrenia. We have profiled some of the preclinical biochemical, electrophysiological, and behavioral consequences of administering potent and selective inhibitors of DAAO to rodents to begin to test this hypothesis. Inhibition of DAAO activity resulted in a significant dose and time dependent increase in D-serine only in the cerebellum, although a time delay was observed between peak plasma or brain drug concentration and cerebellum D-serine response. Pharmacokinetic/pharmacodynamic (PK/PD) modeling employing a mechanism-based indirect response model was used to characterize the correlation between free brain drug concentration and D-serine accumulation. DAAO inhibitors had little or no activity in rodent models considered predictive for antipsychotic activity. The inhibitors did, however, affect cortical activity in the Mescaline-Induced Scratching model, produced a modest but significant increase in NMDA receptor-mediated synaptic currents in primary neuronal cultures from rat hippocampus, and resulted in a significant increase in evoked hippocampal theta rhythm, an in vivo electrophysiological model of hippocampal activity. These findings demonstrate that although DAAO inhibition did not cause a measurable increase in D-serine in forebrain, it did affect hippocampal and cortical activity, possibly through augmentation of NMDA receptor-mediated currents.

α4ß2 nicotinic acetylcholine receptor partial agonists with low intrinsic efficacy have antidepressant-like properties.

Previous studies have suggested that treatment with antagonists or partial agonists of nicotinic acetylcholine receptors containing the ß2-subunit (ß2 nAChRs) results in antidepressant-like effects. In this study, we tested three novel compounds with different affinity and functional efficacy at α4ß2 nAChRs, which were synthesized as part of nAChR discovery projects at Pfizer, in the tail-suspension, forced-swim, and novelty-suppressed feeding tests of antidepressant efficacy. All compounds tested reduced immobility in the forced-swim test and one of the compounds also reduced immobility in the tail-suspension test. All the compounds appeared to affect food intake on their own, with two compounds reducing feeding significantly in the home cage, precluding a clear interpretation of the results in the novelty-suppressed feeding test. None of the compounds altered locomotor activity at the doses and time points used here. Therefore, a subset of these compounds has pharmacological and behavioral properties that demonstrate the potential of nicotinic compounds as a treatment of mood disorders. Further development of nicotinic-based antidepressants should focus on increasing nAChR subtype selectivity to obtain consistent antidepressant properties with an acceptable side-effect profile.

Amelioration of ketamine-induced working memory deficits by dopamine D1 receptor agonists.

RATIONALE: Ketamine has been used in humans to model cardinal symptoms of schizophrenia, including working memory impairments and behavioral disorganization. Translational studies with ketamine in nonhuman primates promise to extend the neurobiological understanding of this model. OBJECTIVES: By establishing the dose-dependent effects of ketamine on spatial working memory and behavior, we sought to test and compare the capacity of antipsychotic and procognitive agents to reverse these symptoms. METHODS: Behavioral observations were taken following administration of placebo/ketamine (0.1-1.7 mg/kg, intramuscularly) and animals were tested on the spatial delayed response task 15 min post-injection. Pretreatments with risperidone as well as full and partial D1 receptor agonists were tested for their ability to reverse ketamine-induced impairments. RESULTS: Ketamine (median 1.0 mg/kg) produced a profound cognitive impairment and behavioral sequelae reminiscent of positive and negative symptoms. Risperidone within the therapeutic dose range failed to antagonize behavioral or cognitive consequences of acute ketamine but A77636 (0.1 and 1 microg/kg) and SKF38393 (0.1 microg/kg-100 microg/kg) ameliorated the spatial working memory deficit. This effect of A77636 was blocked by the D1 receptor antagonist, SCH39166 (1 and 10 microg/kg). CONCLUSIONS: These findings establish a valuable ketamine platform relevant to the treatment of cognitive dysfunction in schizophrenia. The reversal of ketamine-induced working memory deficits by a D1 receptor agonist, but not a commonly prescribed atypical antipsychotic, provides behavioral evidence for significant D1/N-methyl-D: -aspartate receptor interactions in prefrontal dysfunction and concurs with suggestions that D1 agonists may be useful in the treatment of cognitive impairments in schizophrenia.

Prevention of ketamine-induced working memory impairments by AMPA potentiators in a nonhuman primate model of cognitive dysfunction.

Working memory impairments are a core aspect of schizophrenia, yet current medicines do not address such cognitive dysfunction. We have developed a model of these working memory deficits by acutely disrupting glutamatergic synaptic transmission by administration of the N-methyl-d-aspartate (NMDA) antagonist ketamine in the nonhuman primate. The current studies evaluated the effect of positive allosteric modulators ("potentiators") of the alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA) receptors on the working memory and behavioral effects of ketamine. AMPA receptors mediate fast excitatory synaptic transmission throughout the brain and play a critical role in the activity-dependent regulation of NMDA receptors. We find that positive modulation of AMPA receptors with LY451646 (0.1-1.0mg/kg, SC) and structurally distinct PF-4778574 (0.01mg/kg, SC) robustly ameliorates ketamine-induced working memory impairments without altering behavioral effects of acute ketamine we consider related to positive- and negative-like symptoms. These results support AMPA receptor potentiators as a potential adjunctive treatment for cognitive impairment associated with schizophrenia (CIAS).

Glycine transporter inhibition reverses ketamine-induced working memory deficits.

Glycine transporter inhibitors have recently been reported to improve symptoms in patients with schizophrenia. Here we used acute ketamine in the nonhuman primate to test the effectiveness of the novel glycine transporter inhibitor, PF-3463275, in a model of cognitive dysfunction relevant to schizophrenia. PF-3463275 (0.01-0.17 mg/kg; subcutaneously) or a vehicle was given before the administration of ketamine (median dose of 1.0 mg/kg intramuscularly) or placebo (saline). Ketamine induced hallucinatory-like behaviors that were not reversed by PF-3463275. In contrast, all doses of PF-3463275 alleviated the deficit in spatial working memory induced by ketamine. Theses findings build upon those in patients by providing translational support for targeting glycine transporter in adjunctive treatment for cognitive dysfunction in schizophrenia.

Preclinical pharmacology of the alpha4beta2 nAChR partial agonist varenicline related to effects on reward, mood and cognition.

The pharmacological properties and pharmacokinetic profile of the alpha4beta2 nicotinic acetylcholine receptor (nAChR) partial agonist varenicline provide an advantageous combination of free brain levels and functional potencies at the target receptor that for a large part explain its efficacy as a smoking cessation aid. Since alpha4beta2 and other nAChR subtypes play important roles in mediating central processes that control reward, mood, cognition and attention, there is interest in examining the effects of selective nAChR ligands such as varenicline in preclinical animal models that assess these behaviors. Here we describe results from studies on varenicline's effects in animal models of addiction, depression, cognition and attention and discuss these in the context of recently published preclinical and preliminary clinical studies that collected data on varenicline's effects on mood, cognition and alcohol abuse disorder. Taken together, the preclinical and the limited clinical data show beneficial effects of varenicline, but further clinical studies are needed to evaluate whether the preclinical effects observed in animal models are translatable to the clinic.

Varenicline has antidepressant-like activity in the forced swim test and augments sertraline's effect.

Varenicline, an alpha4beta2 nicotinic acetylcholine receptor partial agonist developed as a smoking cessation aid, showed antidepressant-like activity in the forced swim test in two mouse strains. In addition, a low varenicline dose significantly enhanced the effects of moderately active doses of the selective serotonin reuptake inhibitor sertraline. These findings are consistent with the notion that reducing alpha4beta2 nicotinic acetylcholine receptor activity either by antagonists or by partial agonists that can partially activate or desensitize acetylcholine receptors is associated with antidepressant-like properties. These data suggest that varenicline may have antidepressant potential and can, when combined, augment antidepressant responses of selective serotonin reuptake inhibitors.

CP-809,101, a selective 5-HT2C agonist, shows activity in animal models of antipsychotic activity.

CP-809,101 is a potent, functionally selective 5-HT(2C) agonist that displays approximately 100% efficacy in vitro. The aim of the present studies was to assess the efficacy of a selective 5-HT(2C) agonist in animal models predictive of antipsychotic-like efficacy and side-effect liability. Similar to currently available antipsychotic drugs, CP-809,101 dose-dependently inhibited conditioned avoidance responding (CAR, ED(50)=4.8 mg/kg, sc). The efficacy of CP-809,101 in CAR was completely antagonized by the concurrent administration of the 5-HT(2C) receptor antagonist, SB-224,282. CP-809,101 antagonized both PCP- and d-amphetamine-induced hyperactivity with ED(50) values of 2.4 and 2.9 mg/kg (sc), respectively and also reversed an apomorphine induced-deficit in prepulse inhibition. At doses up to 56 mg/kg, CP-809,101 did not produce catalepsy. Thus, the present results demonstrate that the 5-HT(2C) agonist, CP-809,101, has a pharmacological profile similar to that of the atypical antipsychotics with low extrapyramidal symptom liability. CP-809,101 was inactive in two animal models of antidepressant-like activity, the forced swim test and learned helplessness. However, CP-809,101 was active in novel object recognition, an animal model of cognitive function. These data suggest that 5-HT(2C) agonists may be a novel approach in the treatment of psychosis as well as for the improvement of cognitive dysfunction associated with schizophrenia.

Corticotropin-releasing factor receptor subtypes mediating nutritional suppression of estrous behavior in Syrian hamsters.

Caloric deprivation inhibits reproduction, including copulatory behaviors, in female mammals. Decreases in metabolic fuel availability are detected in the hindbrain, and this information is relayed to the forebrain circuits controlling estrous behavior by neuropeptide Y (NPY) projections. In the forebrain, the nutritional inhibition of estrous behavior appears to be mediated by corticotropin-releasing factor (CRF) or urocortin-signaling systems. Intracerebroventricular (ICV) infusion of the CRF antagonist, astressin, prevents the suppression of lordosis by food deprivation and by NPY treatment in Syrian hamsters. These experiments sought to determine which CRF receptor type(s) is involved. ICV infusion of the CRF receptor subtype CRFR2-selective agonists urocortin 2 and 3 (UCN2, UCN3) inhibited sexual receptivity in hormone-primed, ovariectomized hamsters. Furthermore, the CRFR2-selective antagonist, astressin 2B, prevented the inhibition of estrous behavior by UCN2 and by NPY, consistent with a role for CRFR2. On the other hand, astressin 2B did not prevent the inhibition of behavior induced by 48-h food deprivation or ICV administration of CRF, a mixed CRFR1 and CRFR2 agonist, suggesting that activation of CRFR1 signaling is sufficient to inhibit sexual receptivity in hamsters. Although administration of CRFR1-selective antagonists (NBI-27914 and CP-154,526) failed to reverse the inhibition of receptivity by CRF treatment, we could not confirm their biological effectiveness in hamsters. The most parsimonious interpretation of these findings is that, although NPY inhibits estrous behavior via downstream CRFR2 signaling, food deprivation may exert its inhibition via both CRFR1 and CRFR2 and that redundant neuropeptide systems may be involved.

The pharmacology of CP-154,526, a non-peptide antagonist of the CRH1 receptor: a review.

Since CRH has been shown to mediate stress-induced physiological and behavioral changes, it has been hypothesized that CRH receptor antagonists may have therapeutic potential in disorders that involve excessive CRH activity. CP-154,526 and its close analog antalarmin are potent, brain-penetrable, selective nonpeptide CRH1 receptor antagonists that were discovered in an effort to develop compounds with efficacy in CNS disorders precipitated by stress. Since its discovery many investigators have used CP-154,526 as a tool to study the pharmacology of CRH and its receptors and to evaluate its therapeutic potential in a variety of CNS and peripheral disorders. Systemically-administered CP-154,526 has been demonstrated to antagonize CRH- and stress-induced neuroendocrine, neurochemical, electrophysiological, and behavioral effects. These findings support the hypothesis that CRH1 receptor antagonists may have therapeutic utility in a number of neuropsychiatric disorders. CP-154,526, as well as other CRH1 receptor antagonists that have since been discovered, have also shown activity in several preclinical models of anxiety, depression, and substance abuse, while having little effect on locomotor activity and motor function. Although these effects are on occasion inconsistent among different laboratories, clinical evaluation of CRH1 antagonists appears justified on the basis of these and clinical data implicating the involvement of CRH in several CNS disorders. The effects of CRH1 antagonists on cognition, neurodegeneration, inflammation, and the gastrointestinal system have not been as extensively characterized and additional studies will be necessary to evaluate their therapeutic potential in these areas.

CP-465,022, a selective noncompetitive AMPA receptor antagonist, blocks AMPA receptors but is not neuroprotective in vivo.

BACKGROUND AND PURPOSE: Alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor inhibition has been hypothesized to provide neuroprotective efficacy after cerebral ischemia on the basis of the activity in experimental ischemia models of a variety of compounds with varying selectivity for AMPA over other glutamate receptor subtypes. CP-465,022 is a new, potent, and selective noncompetitive AMPA receptor antagonist. The present study investigated the ability of this compound to reduce neuronal loss after experimental cerebral ischemia to probe the neuroprotective potential of AMPA receptor inhibition. METHODS: To demonstrate that CP-465,022 gains access to the brain, the effects of systemic administration of CP-465,022 were investigated on AMPA receptor-mediated electrophysiological responses in hippocampus and on chemically induced seizures in rats. The compound was then investigated for neuroprotective efficacy in rat global and focal ischemia models at doses demonstrated to be maximally effective in the electrophysiology and seizure models. RESULTS: CP-465,022 potently and efficaciously inhibited AMPA receptor-mediated hippocampal synaptic transmission and the induction of seizures. However, at comparable doses, CP-465,022 failed to prevent CA1 neuron loss after brief global ischemia or to reduce infarct volume after temporary middle cerebral artery occlusion. CONCLUSIONS: Given the high selectivity of CP-465,022 for AMPA over kainate and N-methyl-D-aspartate subtypes of glutamate receptors, the lack of neuroprotective efficacy of the compound calls into question the neuroprotective efficacy of AMPA receptor inhibition after ischemia.

Quest for knowledge. Is clinical research right for your practice?

Ten considerations medical groups should ponder before they agree to participate in clinical research. Staff time, patient well-being, and data integrity top the list of concerns.