The benefits, limitations and opportunities of preclinical models for neonatal drug development.

Increased research to improve preclinical models to inform the development of therapeutics for neonatal diseases is an area of great need. This article reviews five common neonatal diseases - bronchopulmonary dysplasia, retinopathy of prematurity, necrotizing enterocolitis, perinatal hypoxic-ischemic encephalopathy and neonatal sepsis - and the available in vivo, in vitro and in silico preclinical models for studying these diseases. Better understanding of the strengths and weaknesses of specialized neonatal disease models will help to improve their utility, may add to the understanding of the mode of action and efficacy of a therapeutic, and/or may improve the understanding of the disease pathology to aid in identification of new therapeutic targets. Although the diseases covered in this article are diverse and require specific approaches, several high-level, overarching key lessons can be learned by evaluating the strengths, weaknesses and gaps in the available models. This Review is intended to help guide current and future researchers toward successful development of therapeutics in these areas of high unmet medical need.

An Industry Survey With Focus on Cardiovascular Safety Pharmacology Study Design and Data Interpretation.

INTRODUCTION: The Safety Pharmacology Society (SPS) conducted a membership survey to examine industry practices related mainly to cardiovascular (CV) safety pharmacology (SP). METHODS: Questions addressed nonclinical study design, data analysis methods, drug-induced effects, and conventional and novel CV assays. RESULTS: The most frequent therapeutic area targeted by drugs developed by the companies/institutions that employ survey responders was oncology. The most frequently observed drug-mediated effects included an increased heart rate, increased arterial blood pressure, hERG (IKr) block, decreased arterial blood pressure, decreased heart rate, QTc prolongation, and changes in body temperature. Broadly implemented study practices included Latin square crossover study design with n = 4 for nonrodent CV studies, statistical analysis of data (eg, analysis of variance), use of arrhythmia detection software, and the inclusion of data from all study animals when integrating SP studies into toxicology studies. Most responders frequently used individual animal housing conditions. Responders commonly evaluated drug effects on multiple ion channels, but in silico modeling methods were used much less frequently. Most responders rarely measured the J-Tpeak interval in CV studies. Uncertainties relative to Standard for Exchange of Nonclinical Data applications for data derived from CV SP studies were common. Although available, the use of human induced pluripotent stem cell cardiomyocytes remains rare. The respiratory SP study was rarely involved with identifying drug-induced functional issues. Responders indicated that the study-derived no observed effect level was more frequently determined than the no observed adverse effect level in CV SP studies; however, a large proportion of survey responders used neither.

Trifluoromethyl Dihydrothiazine-Based ß-Secretase (BACE1) Inhibitors with Robust Central ß-Amyloid Reduction and Minimal Covalent Binding Burden.

The ß-site amyloid precursor protein cleaving enzyme 1 (BACE1, also known as ß-secretase) is a promising target for the treatment of Alzheimer's disease. A pKa lowering approach over the initial leads was adopted to mitigate hERG inhibition and P-gp efflux, leading to the design of 6-CF3 dihydrothiazine 8 (N-(3-((4S,6S)-2-amino-4-methyl-6-(trifluoromethyl)-5,6-dihydro-4H-1,3-thiazin-4-yl)-4-fluorophenyl)-5-cyanopicolinamide). Optimization of 8 led to the discovery of 15 (N-(3-((4S,6S)-2-amino-4-methyl-6-(trifluoromethyl)-5,6-dihydro-4H-1,3-thiazin-4-yl)-4-fluorophenyl)-5-(fluoromethoxy)pyrazine-2-carboxamide) with an excellent balance of potency, hERG inhibition, P-gp efflux, and metabolic stability. Oral administration of 8 elicited robust Aß reduction in dog even at 0.16 mg/kg. Reflecting the reduced hERG inhibitory activity, no QTc prolongation was observed at high doses. The potential for reactive metabolite formation of 15 was realized in a nucleophile trapping assay using [14 C]-KCN in human liver microsomes. Utilizing covalent binding (CVB) in human hepatocytes and the maximum projected human dosage, the daily CVB burden of 15 was calculated to be at an acceptable value of below 1 mg/day. However, hepatotoxicity was observed when 15 was subjected to a two-week tolerance study in dog, which prevented further evaluation of this compound.

Discovery of an Extremely Potent Thiazine-Based ß-Secretase Inhibitor with Reduced Cardiovascular and Liver Toxicity at a Low Projected Human Dose.

Genetic evidence points to deposition of amyloid-ß (Aß) as a causal factor for Alzheimer's disease. Aß generation is initiated when ß-secretase (BACE1) cleaves the amyloid precursor protein. Starting with an oxazine lead 1, we describe the discovery of a thiazine-based BACE1 inhibitor 5 with robust Aß reduction in vivo at low concentrations, leading to a low projected human dose of 14 mg/day where 5 achieved sustained Aß reduction of 80% at trough level.

Identification and Optimization of Pyrrolo[3,2-d]pyrimidine Toll-like Receptor 7 (TLR7) Selective Agonists for the Treatment of Hepatitis B.

Pyrrolo[3,2-d]pyrimidines were identified as a new series of potent and selective TLR7 agonists. Compounds were optimized for their activity and selectivity over TLR8. This presents an advantage over recently described scaffolds that have residual TLR8 activity, which may be detrimental to the tolerability of the candidate drug. Oral administration of the lead compound 54 effectively induced a transient interferon stimulated gene (ISG) response in mice and cynomolgus monkeys. We aimed for a high first pass effect, limiting cytokine induction systemically, and demonstrated the potential for the immunotherapy of viral hepatitis.

A multidimensional sight on cardiac failure: uncovered from structural to molecular level.

Heart failure is one of the leading causes of death, with high mortality rate within 5 years after diagnosis. Treatment and prognosis options for heart failure primarily targeted on hemodynamic and neurohumoral components that drive progressive deterioration of the heart. However, given the multifactorial background that eventually leads to the "phenotype" named heart failure, better insight into the various components may lead to personalized treatment opportunities. Indeed, currently used criteria to diagnose and/or classify heart failure are possibly too focused on phenotypic improvement rather than the molecular driver of the disease and could therefore be further refined by integrating the leap of molecular and cellular knowledge. The ambiguity of the ejection fraction-based classification criteria became evident with development of advanced molecular techniques and the dawn of omics disciplines which introduced the idea that disease is caused by a myriad of cellular and molecular processes rather than a single event or pathway. The fact that different signaling pathways may underlie similar clinical manifestations calls for a more holistic study of heart failure. In this context, the systems biology approach can offer a better understanding of how different components of a system are altered during disease and how they interact with each other, potentially leading to improved diagnosis and classification of this condition. This review is aimed at addressing heart failure through a multilayer approach that covers individually some of the anatomical, morphological, functional, and tissue aspects, with focus on cellular and subcellular features as an alternative insight into new therapeutic opportunities.

Single Dose Caffeine Protects the Neonatal Mouse Brain against Hypoxia Ischemia.

In this randomized blinded study, we investigated caffeine 5 mg/kg treatment given directly after neonatal brain hypoxia ischemia. Brain morphology, behavior and key brain infiltrating immune populations were examined. Caffeine treatment significantly improves outcome when compared to phosphate buffered saline. Flow cytometric analysis of immune responses revealed no persistent immunological alterations. Given its safety caffeine emerges as a candidate for neuroprotective intervention after neonatal brain injury.

HCN channelopathy and cardiac electrophysiologic dysfunction in genetic and acquired rat epilepsy models.

OBJECTIVE: Evidence from animal and human studies indicates that epilepsy can affect cardiac function, although the molecular basis of this remains poorly understood. Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels generate pacemaker activity and modulate cellular excitability in the brain and heart, with altered expression and function associated with epilepsy and cardiomyopathies. Whether HCN expression is altered in the heart in association with epilepsy has not been investigated previously. We studied cardiac electrophysiologic properties and HCN channel subunit expression in rat models of genetic generalized epilepsy (Genetic Absence Epilepsy Rats from Strasbourg, GAERS) and acquired temporal lobe epilepsy (post-status epilepticus SE). We hypothesized that the development of epilepsy is associated with altered cardiac electrophysiologic function and altered cardiac HCN channel expression. METHODS: Electrocardiography studies were recorded in vivo in rats and in vitro in isolated hearts. Cardiac HCN channel messenger RNA (mRNA) and protein expression were measured using quantitative PCR and Western blotting respectively. RESULTS: Cardiac electrophysiology was significantly altered in adult GAERS, with slower heart rate, shorter QRS duration, longer QTc interval, and greater standard deviation of RR intervals compared to control rats. In the post-SE model, we observed similar interictal changes in several of these parameters, and we also observed consistent and striking bradycardia associated with the onset of ictal activity. Molecular analysis demonstrated significant reductions in cardiac HCN2 mRNA and protein expression in both models, providing a molecular correlate of these electrophysiologic abnormalities. SIGNIFICANCE: These results demonstrate that ion channelopathies and cardiac dysfunction can develop as a secondary consequence of chronic epilepsy, which may have relevance for the pathophysiology of cardiac dysfunction in patients with epilepsy.

Long lasting local and systemic inflammation after cerebral hypoxic ischemia in newborn mice.

BACKGROUND: Hypoxic ischemia (HI) is an important cause of neonatal brain injury and subsequent inflammation affects neurological outcome. In this study we performed investigations of systemic and local activation states of inflammatory cells from innate and adaptive immunity at different time points after neonatal HI brain injury in mice. METHODOLOGY/PRINCIPAL FINDINGS: We developed a multiplex flow cytometry based method combined with immunohistochemistry to investigate cellular immune responses in the brain 24 h to 7 months after HI brain injury. In addition, functional studies of ex vivo splenocytes after cerebral hypoxic ischemia were performed. Both central and peripheral activation of CD11b(+) and CD11c(+) antigen presenting cells were seen with expression of the costimulatory molecule CD86 and MHC-II, indicating active antigen presentation in the damaged hemisphere and in the spleen. After one week, naïve CD45rb(+) T-lymphocytes were demonstrated in the damaged brain hemisphere. In a second phase after three months, pronounced activation of CD45rb(-) T-lymphocytes expressing CD69 and CD25 was seen in the damaged hemisphere. Brain homogenate induced proliferation in splenocytes after HI but not in controls. CONCLUSIONS/SIGNIFICANCE: Our findings demonstrate activation of both local and systemic immune responses months after hypoxic ischemic neonatal brain injury. The long term immune activation observed is of general importance for future studies of the inflammatory response after brain injury as most previous studies have focused on the first few weeks after damage, while the effects of the late inflammation phase may be missed. Furthermore, the self-reactive component raises the question if there is a correlation with development of autoimmune brain disease later in life.

Cooperative cardioprotection through adenosine A1 and A2A receptor agonism in ischemia-reperfused isolated mouse heart.

Recent reports have shown that adenosine A1 receptor-mediated cardioprotection requires concomitant A2 receptor activation, but no study thus far has shown that this phenomenon occurs using A1 agonists at reperfusion. Thus, we compared adenosine A2A receptor knockout (A2AKO) and wild-type mouse hearts (n = 9-11) subjected to global ischemia (30 minutes) and reperfusion (60 minutes) in the presence and absence of the A1 agonist N-cyclopentlyadenosine (CPA). We also determined the effects of selective antagonists at A2A and A2B receptors on CPA-induced protection. In wild-type hearts, CPA (100 nM) significantly (P < 0.05) improved contractility (52.7 ± 6.2% versus 23.9 ± 4.9% of preischemia), left ventricular developed pressure, end diastolic pressure; reduced infarct size (7.9 ± 1.7% versus 23.9 ± 6.6% area at risk); decreased lactate dehydrogenase efflux; and increased ERK1/2 phosphorylation at 60 minutes of reperfusion. Adenosine A2A (ZM241385, 50 nM) and A2B (MRS1754, 100 nM) receptor antagonists abolished CPA-mediated cardioprotection in wild-type groups as did the A1 receptor antagonist DPCPX (P < 0.05). In A2AKO hearts, CPA did not improve functional parameters and protective signaling with the exception of end diastolic pressure. In this model, using a clinically relevant mode of pharmacologic intervention, pERK 1/2-dependent A1-mediated cardioprotection requires a cooperative activation of A2 receptors, presumably through endogenous adenosine.

A novel highly selective adenosine A1 receptor agonist VCP28 reduces ischemia injury in a cardiac cell line and ischemia-reperfusion injury in isolated rat hearts at concentrations that do not affect heart rate.

The cardioprotective effects of a novel adenosine A1 receptor agonist N6-(2,2,5,5-tetramethylpyrrolidin-1-yloxyl-3-ylmethyl) adenosine (VCP28) were compared with the selective adenosine A1 receptor agonist N6-cyclopentyladenosine (CPA) in a H9c2(2-1) cardiac cell line-simulated ischemia (SI) model (12 hours) and a global ischemia (30 minutes) and reperfusion (60 minutes) model in isolated rat heart model. H9c2(2-1) cells were treated with CPA and VCP28 at the start of ischemia for entire ischemic duration, whereas isolated rat hearts were treated at the onset of reperfusion for 15 minutes. In the H9c2(2-1) cells SI model, CPA and VCP28 (100 nM) significantly (P < 0.05, n = 5-6) reduced the proportion of nonviable cells (30.88% +/- 2.49% and 16.17% +/- 3.77% of SI group, respectively) and lactate dehydrogenase efflux. In isolated rat hearts, CPA and VCP28 significantly (n = 6-8, P < 0.05) improved post-ischemic contractility (dP/dt(max), 81.69% +/- 10.96%, 91.07% +/- 19.87% of baseline, respectively), left ventricular developed pressure, and end diastolic pressure and reduced infarct size. The adenosine A1 receptor antagonist abolished the cardioprotective effects of CPA and VCP28 in SI model and isolated rat hearts. In conclusion, the adenosine A1 receptor agonist VCP28 has equal cardioprotective effects to the prototype A1 agonist CPA at concentrations that have no effect on heart rate.

Delineating the mode of action of adenosine A1 receptor allosteric modulators.

Despite the identification of 2-amino-3-benzoylthiophenes (2A3BTs) as the first example of small-molecule allosteric potentiators of agonist function at a G protein-coupled receptor (GPCR)-the adenosine A(1) receptor-their mechanism of action is still not fully understood. We now report the mechanistic basis for the complex behaviors noted for 2A3BTs at A(1) receptors. Using a combination of membrane-based and intact-cell radioligand binding, multiple signaling assays, and a native tissue bioassay, we found that the allosteric interaction between 2A3BTs and the agonists 2-chloro-N(6)-[(3)H]cyclopentyladenosine or (-)-N(6)-(2-phenylisopropyl)adenosine (R-PIA) or the antagonist [(3)H]8-cyclopentyl-1,3-dipropylxanthine is consistent with a ternary complex model involving recognition of a single extracellular allosteric site. However, when allowed access to the intracellular milieu, 2A3BTs have a secondary action as direct G protein inhibitors; this latter property is receptor-independent as it is observed in nontransfected cells and also after stimulation of another GPCR. In addition, we found that 2A3BTs can signal as allosteric agonists in their own right but show bias toward certain pathways relative to the orthosteric agonist, R-PIA. These results indicate that 2A3BTs have a dual mode of action when interacting with the A(1) receptor and that they can engender novel functional selectivity in A(1) signaling. These mechanisms need to be factored into allosteric ligand structure-activity studies.

Synthesis and evaluation of new N6-substituted adenosine-5'-N-methylcarboxamides as A3 adenosine receptor agonists.

A number of N(6)-substituted adenosine-5'-N-methylcarboxamides were synthesised and their pharmacology, in terms of their receptor affinity, selectivity and cardioprotective effects, were explored. The first series of compounds, 4a-4f and 5a-5f, showed modest receptor affinity for the A(3)AR with K(i) values in the low to mid muM range. However, the incorporation of a 4-(2-aminoethyl)-2,6-di-tert-butylphenol group in the N(6)-position (in compounds 4g and 5g) significantly improved the affinity with K(i) values of 30 and 9 nM, respectively. Improvements in affinity, as well as selectivity were seen when a functionalized linker was introduced. The N(6)-phenyl series, compounds 7a-7d, demonstrated low to mid nanomolar receptor affinities (K(i)=2.3-45.0 nM), with 7b displaying 109-fold selectivity for the A(3)AR (vs A(1)). The N(6)-benzyl series 9a-9c also proved to be potent and selective A(3)AR agonists and the longer chain length linker 13 was tolerated at the A(3)AR without abrogation of affinity or selectivity. Cardioprotection was demonstrated by a simulated ischaemia cell culture assay, whereby 7b, 7c, 9a, 9b and 9c all showed cardioprotective effects at 100 nM comparable or better than the benchmark A(3)AR agonist IB-MECA, but which were indistinguishable by statistical analysis. For example, compound 9c reduced cell death by 68.0+/-3.6%.

Cardioprotection induced by adenosine A1 receptor agonists in a cardiac cell ischemia model involves cooperative activation of adenosine A2A and A2B receptors by endogenous adenosine.

Extracellular adenosine concentrations increase within the heart during ischemia, and any exogenous adenosine receptor agonists therefore work in the context of significant local agonist concentrations. We evaluated the interactions between A1, A2A, A2B, and A3 receptors in the presence and absence of adenosine deaminase (ADA, which is used to remove endogenous adenosine) in a cardiac cell ischemia model. Simulated ischemia (SI) was induced by incubating H9c2(2-1) cells in SI medium for 12 hours in 100% N2 gas before assessment of necrosis using propidium iodide (5 microM) or apoptosis using AnnexinV-PE flow cytometry. N6-Cyclopentyladenosine (CPA; 10(-7)M) and N6-(3-iodobenzyl) adenosine-5'-N-methyluronamide (IB-MECA; 10(-7)M) reduced the proportion of nonviable cells to 30.87 +/- 2.49% and 35.18 +/- 10.30%, respectively (% of SI group). In the presence of ADA, the protective effect of CPA was reduced (62.82 +/- 3.52% nonviable), whereas the efficacy of IB-MECA was unchanged (35.81 +/- 3.84% nonviable; P < 0.05, n = 3-5, SI vs. SI + ADA). The protective effects of CPA and IB-MECA were abrogated in the presence of their respective antagonists DPCPX (8-cyclopentyl-1,3-dipropylxanthine) and MRS1191 [3-ethyl-5-benzyl-2-methyl-4-phenylethynyl-6-phenyl-1,4-(+/-)-dihydropyridine-3,5-dicarboxylate], whereas A2A and A2B agonists had no significant effect. CPA-mediated protection was abrogated in the presence of both A2A (ZM241385, 4-(2-[7-amino-2-(2-furyl)[1,2,4]triazolo[2,3-a][1,3,5]triazin-5-lamino]ethyl)phenol; 50 nM) and A2B (MRS1754, 8-[4-[((4-cyanophenyl)carbamoylmethyl)oxy]phenyl]-1,3-di(n-propyl)xanthine; 200 nM) antagonists (n = 3-5, P < 0.05). In the absence of endogenous adenosine, significant protection was observed with CPA in presence of CGS21680 (4-[2-[[6-amino-9-(N-ethyl-b-D-ribofuranuronamidosyl)-9H-purin-2-yl]amino]ethyl]benzenepropanoic acid) or LUF5834 [2-amino-4-(4-hydroxyphenyl)-6-(1H-imidazol-2-ylmethylsulfanyl)pyridine-3,5-dicarbonitrile] (P < 0.05 vs. SI + ADA + CPA). Apoptosis (14.35 +/- 0.15% of cells in SI + ADA group; P < 0.05 vs. control) was not significantly reduced by CPA or IB-MECA. In conclusion, endogenous adenosine makes a significant contribution to A1 agonist-mediated prevention of necrosis in this SI model by cooperative interactions with both A2A and A2B receptors but does not play a role in A3 agonist-mediated protection.

Dual acting antioxidant A1 adenosine receptor agonists.

Herein we report the synthesis and biological evaluation of some potent and selective A(1) adenosine receptor agonists, which incorporate a functionalised linker attached to an antioxidant moiety. N(6)-(2,2,5,5-Tetramethylpyrrolidin-1-yloxyl-3-ylmethyl)adenosine (VCP28, 2e) proved to be an agonist with high affinity (K(i)=50nM) and good selectivity (A(3)/A(1) > or = 400) for the A(1) adenosine receptor. N(6)-[4-[2-[1,1,3,3-Tetramethylisoindolin-2-yloxyl-5-amido]ethyl]phenyl]adenosine (VCP102, 5a) has higher binding affinity (K(i)=7 nM), but lower selectivity (A(3)/A(1)= approximately 3). All compounds bind weakly (K(i)>1 microM) to A(2A) and A(2B) receptors. The combination of A(1) agonist activity and antioxidant activity has the potential to produce cardioprotective effects.