Basolateral amygdala corticotropin releasing factor receptor 2 interacts with nonmuscle myosin II to destabilize memory in males.

Preclinical studies show that inhibiting the actin motor ATPase nonmuscle myosin II (NMII) with blebbistatin (Blebb) in the basolateral amgydala (BLA) depolymerizes actin, resulting in an immediate, retrieval-independent disruption of methamphetamine (METH)-associated memory in male and female adult and adolescent rodents. The effect is highly selective, as NMII inhibition has no effect in other relevant brain regions (e.g., dorsal hippocampus [dPHC], nucleus accumbens [NAc]), nor does it interfere with associations for other aversive or appetitive stimuli, including cocaine (COC). To understand the mechanisms responsible for drug specific selectivity we began by investigating, in male mice, the pharmacokinetic differences in METH and COC brain exposure . Replicating METH's longer half-life with COC did not render the COC association susceptible to disruption by NMII inhibition. Therefore, we next assessed transcriptional differences. Comparative RNA-seq profiling in the BLA, dHPC and NAc following METH or COC conditioning identified crhr2, which encodes the corticotropin releasing factor receptor 2 (CRF2), as uniquely upregulated by METH in the BLA. CRF2 antagonism with Astressin-2B (AS2B) had no effect on METH-associated memory after consolidation, allowing for determination of CRF2 influences on NMII-based susceptibility. Pretreatment with AS2B prevented the ability of Blebb to disrupt an established METH-associated memory. Alternatively, combining CRF2 overexpression and agonist treatment, urocortin 3 (UCN3), in the BLA during conditioning rendered COC-associated memory susceptible to disruption by NMII inhibition, mimicking the Blebb-induced, retrieval-independent memory disruption seen with METH. These results suggest that BLA CRF2 receptor activation during memory formation in male mice can prevent stabilization of the actin-myosin cytoskeleton supporting the memory, rendering it vulnerable to disruption by NMII inhibition. CRF2 represents an interesting target for BLA-dependent memory destabilization via downstream effects on NMII.

Basolateral Amygdala Corticotrophin Releasing Factor Receptor 2 Interacts with Nonmuscle Myosin II to Destabilize Memory.

Inhibiting the actin motor ATPase nonmuscle myosin II (NMII) with blebbistatin (Blebb) in the basolateral amgydala (BLA) depolymerizes actin, resulting in an immediate, retrieval-independent disruption of methamphetamine (METH)-associated memory. The effect is highly selective, as NMII inhibition has no effect in other relevant brain regions (e.g. dorsal hippocampus [dPHC], nucleus accumbens [NAc]), nor does it interfere with associations for other aversive or appetitive stimuli, including cocaine (COC). To investigate a potential source of this specificity, pharmacokinetic differences in METH and COC brain exposure were examined. Replicating METH's longer half-life with COC did not render the COC association susceptible to disruption by NMII inhibition. Therefore, transcriptional differences were next assessed. Comparative RNA-seq profiling in the BLA, dHPC and NAc following METH or COC conditioning identified crhr2, which encodes the corticotrophin releasing factor receptor 2 (CRF2), as uniquely upregulated by METH in the BLA. CRF2 antagonism with Astressin-2B (AS2B) had no effect on METH-associated memory after consolidation, allowing for determination of CRF2 influences on NMII-based susceptibility after METH conditioning. Pretreatment with AS2B occluded the ability of Blebb to disrupt an established METH-associated memory. Alternatively, the Blebb-induced, retrieval-independent memory disruption seen with METH was mimicked for COC when combined with CRF2 overexpression in the BLA and its ligand, UCN3 during conditioning. These results indicate that BLA CRF2 receptor activation during learning can prevent stabilization of the actin-myosin cytoskeleton supporting the memory, rendering it vulnerable to disruption via NMII inhibition. CRF2 represents an interesting target for BLA-dependent memory destabilization via downstream effects on NMII.

Addressing the oxamniquine in vitro-in vivo paradox to facilitate a new generation of anti-schistosome treatments.

The antischistosomal drug oxamniquine, OXA, requires activation by a sulfotransferase within the parasitic worm to enable killing. Examination of the pharmacokinetic/pharmacodynamic (PK/PD) relationship for OXA identified an in vitro-in vivo paradox with the maximal clinical plasma concentrations five-to ten-times lower than the efficacious concentration for in vitro schistosomal killing. The parasite resides in the vasculature between the intestine and the liver, and modeling the PK data to determine portal concentrations fits with in vitro studies and explains the required human dose. In silico models were used to predict murine dosing to recapitulate human conditions for OXA portal concentration and time course. Follow-up PK studies verified in mice that a 50-100 mg/kg oral gavage dose of OXA formulated in acetate buffer recapitulates the 20-40 mg/kg dose common in patients. OXA was rapidly cleared through a combination of metabolism and excretion into bile. OXA absorbance and tissue distribution were similar in wild-type and P-gp efflux transporter knockout mice. The incorporation of in vitro efficacy data and portal concentration was demonstrated for an improved OXA-inspired analog that has been shown to kill S. mansoni, S. haematobium, and S. japonicum, whereas OXA is only effective against S. mansoni. Second-generation OXA analogs should optimize both in vitro killing and physiochemical properties to achieve high portal concentration via rapid oral absorption, facilitated by favorable solubility, permeability, and minimal intestinal metabolism.

A promising chemical series of positive allosteric modulators of the µ-opioid receptor that enhance the antinociceptive efficacy of opioids but not their adverse effects.

Positive allosteric modulators (PAMs) of the µ-opioid receptor (MOR) have been proposed to exhibit therapeutic potential by maximizing the analgesic properties of clinically used opioid drugs while limiting their adverse effects or risk of overdose as a result of using lower drug doses. We herein report in vitro and in vivo characterization of two small molecules from a chemical series of MOR PAMs that exhibit: (i) MOR PAM activity and receptor subtype selectivity in vitro, (ii) a differential potentiation of the antinociceptive effect of oxycodone, morphine, and methadone in mouse models of pain that roughly correlates with in vitro activity, and (iii) a lack of potentiation of adverse effects associated with opioid administration, such as somatic withdrawal, respiratory depression, and analgesic tolerance. This series of MOR PAMs holds promise for the development of adjuncts to opioid therapy to mitigate against overdose and opioid use disorders.

A single-arm feasibility cohort study of rivaroxaban in antiphospholipid syndrome.

BACKGROUND: There is uncertainty regarding the safety and effectiveness of direct oral anticoagulant agents in patients with antiphospholipid syndrome (APS). We performed a multicenter feasibility study to examine our ability to identify and obtain consent from eligible APS patients and to obtain 95% adherence with daily rivaroxaban administration, in order to inform and power a larger study. Clinical outcomes of bleeding and thrombosis were also collected. METHODS: APS patients with prior venous thromboembolism (VTE) were recruited over 2 years (Oct 2014-Sept 2016) and followed for ≥ 1 year. Patients were assessed clinically every 3 months and had pill counts performed every 6 months. Numbers of patients fulfilling study criteria, as well as those consenting to participate, were tracked, and percentage adherence based on pill counts was recorded. These data were compared against the feasibility endpoints. Rates of thrombosis and bleeding were calculated. Criterion for feasibility was obtaining consent from 135 of 150 identified APS patients over 2 years. RESULTS: Ninety-six eligible patients were identified, and 14 declined participation. Eighty-two patients were followed for a mean of 19 months, representing 129.8 patient-years. Average rivaroxaban adherence was 95.0%. During follow-up, there were 4 thromboembolic events (2 cerebrovascular and 2 VTE). There were no episodes of major bleeding. CONCLUSIONS: Adequately powered comparative trials using patient-important outcomes in APS are unlikely to be successful due to inability to recruit sufficient numbers of study subjects. This study does not reveal a higher than expected risk of recurrent thromboembolic disease compared to historical cohorts; however, this is an uncontrolled study in relatively low-risk APS patients. TRIAL REGISTRATION: The study was registered with clinicaltrials.gov, identifier NCT02116036, April 16, 2014.

Albumin-Oxanorbornadiene Conjugates Formed ex Vivo for the Extended Circulation of Hydrophilic Cargo.

Oxanorbornadiene dicarboxylate (OND) reagents were explored for the purpose of binding and releasing chemical cargos from endogenous circulating serum albumins. ONDs bearing gadolinium chelates as model cargos exhibited variable conjugation efficiencies with albumin in rat subjects that are consistent with the observed reactivity of each linker and their observed stability toward serum hydrolases in vitro. The terminal elimination rate from circulation was dependent on the identity of the OND used, and increased circulation time of gadolinium cargo was achieved for linkers bearing electrophilic fragments designed to react with cysteine-34 of circulating serum albumin. This binding of and release from endogenous albumin highlights the potential of OND linkers in the context of optimizing the pharmacokinetic parameters of drugs or diagnostic agents.

Design, Synthesis, and Biological Evaluation of Indole Biphenylcarboxylic Acids as PPARγ Antagonists.

The thiazolidinediones (TZD) typified by rosiglitazone are the only approved therapeutics targeting PPARγ for the treatment of type-2 diabetes (T2DM). Unfortunately, despite robust insulin sensitizing properties, they are accompanied by a number of severe side effects including congestive heart failure, edema, weight gain, and osteoporosis. We recently identified PPARγ antagonists that bind reversibly with high affinity but do not induce transactivation of the receptor, yet they act as insulin sensitizers in mouse models of diabetes (SR1664).1 This Letter details our synthetic exploration around this novel series of PPARγ antagonists based on an N-biphenylmethylindole scaffold. Structure-activity relationship studies led to the identification of compound 46 as a high affinity PPARγ antagonist that exhibits antidiabetic properties following oral administration in diet-induced obese mice.

Antiobesity Effect of a Small Molecule Repressor of RORγ.

The orphan nuclear receptor RORγ is a key regulator for T helper 17 (TH17) cell differentiation, which regulates metabolic and circadian rhythm genes in peripheral tissues. Previously, it was shown that the small molecule inverse agonist of RORγ SR1555 [1-(4-((4'-(1,1,1,3,3,3-hexafluoro-2-hydroxypropan-2-yl)-[1,1'-biphenyl]-4-yl)methyl)piperazin-1-yl) ethanone] suppressed TH17 differentiation and stimulated induced T regulatory (iTreg) cells. Here, we show that treatment of cultured pre-adipocyctes with SR1555 represses the expression of RORγ while leading to increased expression of FGF21 and adipoQ. Chronic administration of SR1555 to obese diabetic mice resulted in a modest reduction in food intake accompanied with significant reduction in fat mass, resulting in reduced body weight and improved insulin sensitivity. Analysis ex vivo of treated mice demonstrates that SR1555 induced expression of the thermogenic gene program in fat depots. Further studies in cultured cells showed that SR1555 inhibited activation of hormone-sensitive lipase and increased fatty acid oxidation. Combined, these results suggest that pharmacological repression of RORγ may represent a strategy for treatment of obesity by increasing thermogenesis and fatty acid oxidation, while inhibition of hormone-sensitive lipase activity results in a reduction of serum free fatty acids, leading to improved peripheral insulin sensitivity.

4-Aminopyridyl-based CYP51 inhibitors as anti-Trypanosoma cruzi drug leads with improved pharmacokinetic profile and in vivo potency.

CYP51 is a P450 enzyme involved in the biosynthesis of the sterol components of eukaryotic cell membranes. CYP51 inhibitors have been developed to treat infections caused by fungi, and more recently the protozoan parasite Trypanosoma cruzi, the causative agent of Chagas disease. To specifically optimize drug candidates for T. cruzi CYP51 (TcCYP51), we explored the structure-activity relationship (SAR) of a N-indolyl-oxopyridinyl-4-aminopropanyl-based scaffold originally identified in a target-based screen. This scaffold evolved via medicinal chemistry to yield orally bioavailable leads with potent anti-T. cruzi activity in vivo. Using an animal model of infection with a transgenic T. cruzi Y luc strain expressing firefly luciferase, we prioritized the biaryl and N-arylpiperazine analogues by oral bioavailability and potency. The drug-target complexes for both scaffold variants were characterized by X-ray structure analysis. Optimization of both binding mode and pharmacokinetic properties of these compounds led to potent inhibitors against experimental T. cruzi infection.

Synthesis and biological evaluation of urea derivatives as highly potent and selective rho kinase inhibitors.

RhoA and its downstream effector ROCK mediate stress fiber formation and cell contraction through their effects on the phosphorylation of myosin light chain (MLC). Inhibition of the RhoA/ROCK pathway has proven to be a promising strategy for several indications such as cardiovascular disease, glaucoma, and inflammatory disease. In 2010, our group reported urea-based ROCK inhibitors as potential antiglaucoma agents. These compounds showed potent IC50 values in enzymatic and cell-based assays and significant intraocular pressure (IOP)-lowering effects in rats (∼7 mmHg). (22) To develop more advanced ROCK inhibitors targeting various potential applications (such as myocardial infarction, erectile dysfunction, multiple sclerosis, etc.) in addition to glaucoma, a thorough SAR for this urea-based scaffold was studied. The detailed optimization process, counter-screening, and in vitro and in vivo DMPK studies are discussed. Potent and selective ROCK inhibitors with various in vivo pharmacokinetic properties were discovered.

JNK Inhibition Protects Dopamine Neurons and Provides Behavioral Improvement in a Rat 6-hydroxydopamine Model of Parkinson's Disease.

Parkinson's disease (PD) results from the loss of dopamine neurons located in the substantia nigra pars compacta (SNpc) that project to the striatum. A therapeutic has yet to be identified that halts this neurodegenerative process, and as such, development of a brain penetrant small molecule neuroprotective agent would represent a significant advancement in the treatment of the disease. To fill this void we developed an aminopyrimidine JNK inhibitor (SR-3306) that reduced the loss of dopaminergic cell bodies in the SNpc and their terminals in the striatum produced by unilateral injection of 6-hydroxydopamine (6-OHDA) into the nigrostriatal pathway. Administration of SR-3306 [10 mg/kg/day (s.c.) for 14 days] increased the number of tyrosine hydroxylase immunoreactive (TH(+)) neurons in the SNpc by six-fold and reduced the loss of the TH(+) terminals in the striatum relative to the corresponding side of 6-OHDA-lesioned rats that received only vehicle (p<0.05). In addition, SR-3306 [10 mg/kg/day (s.c.) for 14 days] decreased d-amphetamine-induced circling by 87% compared to 6-OHDA-lesioned animals given vehicle. Steady-state brain levels of SR-3306 at day 14 were 347 nM, which was approximately two-fold higher than the cell-based IC(50) for this compound. Finally, immunohistochemical staining for phospho-c-jun (p-c-jun) revealed that SR-3306 [10 mg/kg/day (s.c.) for 14 days] produced a 2.3-fold reduction of the number of immunoreactive neurons in the SNpc relative to vehicle treated rats. Collectively, these data suggest that orally bioavailable JNK inhibitors may be useful neuroprotective agents for the treatment of Parkinson's disease.

Apelin protects against angiotensin II-induced cardiovascular fibrosis and decreases plasminogen activator inhibitor type-1 production.

OBJECTIVE: To test the hypothesis that apelin protects against angiotensin II (Ang II)-induced cardiovascular fibrosis and vascular remodeling. METHODS AND RESULTS: Wild-type mice administered apelin or apelin along with Ang II exhibited less cardiovascular fibrosis and decreased plasminogen activator inhibitor type-1 (PAI-1) gene expression than mice receiving Ang II, N-nitro-L-arginine methyl ester (L-NAME), apelin plus L-NAME or apelin plus Ang II plus L-NAME. In-vitro analysis using a luciferase construct driven by 3.1 kb of the human PAI-1 promoter revealed that apelin blocked Ang II-mediated PAI-1 gene expression. Immunoblotting for phosphorylated myosin phosphatase subunit and myosin light chain revealed that apelin blocked Ang II activation of the Rho kinase pathway, which is associated with induction of PAI-1 gene expression by Ang II. In addition, treatment of human aortic smooth muscle cells with apelin reduced PAI-1 mRNA and protein production in the presence and absence of Ang II. Conversely, L-NAME treatment attenuated the downregulation of PAI-1 by apelin in cells. CONCLUSION: Apelin protects against cardiac fibrosis and vascular remodeling through direct regulation of PAI-1 gene expression. This protective effect is mediated through the synergistic inhibition of Ang II signaling and increased production of nitric oxide by apelin. Our data extend previous findings and provide new insight into the molecular mechanisms by which apelin elicits a cardioprotective effect.

Synthesis and SAR of novel quinazolines as potent and brain-penetrant c-jun N-terminal kinase (JNK) inhibitors.

Quinazoline 3 was discovered as a novel c-jun N-terminal kinase (JNK) inhibitor with good brain penetration and pharmacokinetic (PK) properties. A number of analogs which were potent both in the biochemical and cellular assays were discovered. Quinazoline 13a was found to be a potent JNK3 inhibitor (IC(50)=40 nM), with >500-fold selectivity over p38, and had good PK and brain penetration properties. With these properties, 13a is considered a potential candidate for in vivo evaluation.

Tetrahydroisoquinoline derivatives as highly selective and potent Rho kinase inhibitors.

Rho kinase (ROCK) is a promising drug target for the treatment of many diseases including hypertension, multiple sclerosis, cancer, and glaucoma. The structure-activity relationships (SAR) around a series of tetrahydroisoquinolines were evaluated utilizing biochemical and cell-based assays to measure ROCK inhibition. These novel ROCK inhibitors possess high potency, high selectivity, and appropriate pharmacokinetic properties for glaucoma applications. The lead compound, 35, had subnanomolar potency in enzyme ROCK-II assays as well as excellent cell-based potency (IC(50) = 51 nM). In a kinase panel profiling, 35 had an off-target hit rate of only 1.6% against 442 kinases. Pharmacology studies showed that compound 35 was efficacious in reducing intraocular pressure (IOP) in rats with reasonably long duration of action. These results suggest that compound 35 may serve as a promising agent for further development in the treatment of glaucoma.

Discovery of Potent and Selective Urea-Based ROCK Inhibitors and Their Effects on Intraocular Pressure in Rats.

A series of urea-based Rho kinase (ROCK) inhibitors were designed and evaluated. The discovered compounds had excellent enzyme and cellular potency, high kinase selectivity, high aqueous solubility, good porcine corneal penetration, and appropriate DMPK profiles for topical applications as antiglaucoma therapeutics.