Evaluation of Longitudinal Pain Study in Sickle Cell Disease (ELIPSIS) by patient-reported outcomes, actigraphy, and biomarkers.

Clinical trials in sickle cell disease (SCD) often focus on health care utilization for painful vaso-occlusive crises (VOCs). However, no objective, quantifiable pain biomarkers exist, pain is not specific to VOCs, health care utilization varies between patients, unreported at-home VOCs likely contribute to long-term outcomes, and patient-reported outcomes are seldom considered. This noninterventional, longitudinal, 6-month study aimed to develop tools to identify VOCs in SCD patients with or without health care utilization. Participants wore an actigraph device, tracking sleep and activity. Patients with SCD used an electronic patient-reported outcome (ePRO) tool to collect data on pain, medication, fatigue, and daily function. Patients self-reported when they experienced VOC pain (VOC day). Biomarkers were collected every 3 weeks (non-VOC). Self-reported VOCs triggered at-home or in-hospital blood collection. The study enrolled 37 participants with SCD; 35 completed the study. Participants reported 114 VOC events and 346 VOC days, of which 62.3% and 78.3%, respectively, were self-treated at home. The ePRO and actigraphy captured end points of pain, functionality, fatigue, activity, and sleep; each was significantly altered on VOC days compared with non-VOC days. Biomarkers collected at home or in the hospital on VOC days were significantly altered compared with non-VOC baseline values, including leukocyte-platelet aggregates, microfluidic-based blood cell adhesion, interleukin-6, C-reactive protein, interleukin-10, tumor necrosis factor-α, and thrombin-antithrombin. The Evaluation of Longitudinal Pain Study in Sickle Cell Disease (ELIPSIS) trial shows the feasibility of accurately monitoring out-of-hospital pain by using patient-reported VOC days as potential end points for clinical trials in SCD; it describes the changes in biomarkers and activity measured by actigraphy that may enable improved identification and assessment of VOCs.

Transcriptional profiling and pathway analysis of CSF-1 and IL-34 effects on human monocyte differentiation.

CSF-1 is the well-known ligand for CSF-1R, which plays a vital role in monocyte-macrophage generation, survival, and function. IL-34 is a newly discovered cytokine that also signals through CSF-1R. Although there are limited data for downstream signaling and pathway activation for CSF-1, none are published, to date, for expression profiles of IL-34. The objective of this study was to characterize and compare the signaling pathways downstream of the CSF-1R receptor, based on these two ligands. This was accomplished through transcriptional profiling and pathway analysis of CD14(+) human monocytes differentiated with each ligand. Additionally, cells were treated with a CSF-1R inhibitor GW2580 to establish that observations associated with each ligand were CSF-1R mediated. Gene expression profiles were generated for each condition using Agilent 4x44K Whole Human Genome Microarrays. Overall profiles generated by each cytokine were similar (~75% of genes) with a dampened effect noted on some pathways (~25% of genes) with IL-34. One key difference observed, between the two cytokines was in the repression of CCR2 message. A similar divergence in protein level was established by FACS analysis. The differential effect on CCR2 expression has major implications for monocyte/macrophage biology including homeostasis and function. Further study of IL-34 effects on monocyte/macrophage biology will shed light on the specific role each ligand plays and the context in which these roles are important. To our knowledge, this study is the first to illustrate downstream transcriptional profiles and pathways of IL-34 in comparison with CSF-1 and identify notable differences in CCR2 expression.

Discovery of potent selective bioavailable phosphodiesterase 2 (PDE2) inhibitors active in an osteoarthritis pain model. Part II: optimization studies and demonstration of in vivo efficacy.

Selective phosphodiesterase 2 (PDE2) inhibitors are shown to have efficacy in a rat model of osteoarthritis (OA) pain. We identified potent, selective PDE2 inhibitors by optimizing residual PDE2 activity in a series of phosphodiesterase 4 (PDE4) inhibitors, while minimizing PDE4 inhibitory activity. These newly designed PDE2 inhibitors bind to the PDE2 enzyme in a cGMP-like binding mode orthogonal to the cAMP-like binding mode found in PDE4. Extensive structure activity relationship studies ultimately led to identification of pyrazolodiazepinone, 22, which was >1000-fold selective for PDE2 over recombinant, full length PDEs 1B, 3A, 3B, 4A, 4B, 4C, 7A, 7B, 8A, 8B, 9, 10 and 11. Compound 22 also retained excellent PDE2 selectivity (241-fold to 419-fold) over the remaining recombinant, full length PDEs, 1A, 4D, 5, and 6. Compound 22 exhibited good pharmacokinetic properties and excellent oral bioavailability (F=78%, rat). In an in vivo rat model of OA pain, compound 22 had significant analgesic activity 1 and 3h after a single, 10 mg/kg, subcutaneous dose.

Discovery of potent, selective, bioavailable phosphodiesterase 2 (PDE2) inhibitors active in an osteoarthritis pain model, part I: transformation of selective pyrazolodiazepinone phosphodiesterase 4 (PDE4) inhibitors into selective PDE2 inhibitors.

We identified potent, selective PDE2 inhibitors by optimizing residual PDE2 activity in a series of PDE4 inhibitors, while simultaneously minimizing PDE4 activity. These newly designed PDE2 inhibitors bind to the PDE2 enzyme in a cGMP-like mode in contrast to the cAMP-like binding mode found in PDE4. Structure activity relationship studies coupled with an inhibitor bound crystal structure in the active site of the catalytic domain of PDE2 identified structural features required to minimize PDE4 inhibition while simultaneously maximizing PDE2 inhibition.

Development and Validation of a Western Blot Method to Quantify Mini-Dystrophin in Human Skeletal Muscle Biopsies.

Duchenne muscular dystrophy (DMD) is a degenerative muscular disease affecting roughly one in 5000 males at birth. The disease is often caused by inherited X-linked recessive pathogenic variants in the dystrophin gene, but may also arise from de novo mutations. Disease-causing variants include nonsense, out of frame deletions or duplications that result in loss of dystrophin protein expression. There is currently no cure for DMD and the few treatment options available aim at slowing muscle degradation. New advances in gene therapy and understanding of dystrophin (DYS) expression in other muscular dystrophies have opened new opportunities for treatment. Therefore, reliable methods are needed to monitor dystrophin expression and assess the efficacy of new therapies for muscular dystrophies such as DMD and Becker muscular dystrophy (BMD). Here, we describe the validation of a novel Western blot (WB) method for the quantitation of mini-dystrophin protein in human skeletal muscle tissues that is easy to adopt in most laboratory settings. This WB method was assessed through precision, accuracy, selectivity, dilution linearity, stability, and repeatability. Based on mini-DYS standard performance, the assay has a dynamic range of 0.5-15 ng protein (per 5 µg total protein per lane), precision of 3.3 to 25.5%, and accuracy of - 7.5 to 3.3%. Our stability assessment showed that the protein is stable after 4 F/T cycles, up to 2 h at RT and after 7 months at - 70°C. Furthermore, our WB method was compared to the results from our recently published LC-MS method. Workflow for our quantitative WB method to determine mini-dystrophin levels in muscle tissues (created in Biorender.com). Step 1 involves protein extraction from skeletal muscle tissue lysates from control, DMD, or BMD biospecimen. Step 2 measures total protein concentrations. Step 3 involves running gel electrophoresis with wild-type dystrophin (wt-DYS) from muscle tissue extracts alongside mini-dystrophin STD curve and mini-DYS and protein normalization with housekeeping GAPDH.

Mechanistic and pharmacological characterization of PF-04457845: a highly potent and selective fatty acid amide hydrolase inhibitor that reduces inflammatory and noninflammatory pain.

The endogenous cannabinoid (endocannabinoid) anandamide is principally degraded by the integral membrane enzyme fatty acid amide hydrolase (FAAH). Pharmacological blockade of FAAH has emerged as a potentially attractive strategy for augmenting endocannabinoid signaling and retaining the beneficial effects of cannabinoid receptor activation, while avoiding the undesirable side effects, such as weight gain and impairments in cognition and motor control, observed with direct cannabinoid receptor 1 agonists. Here, we report the detailed mechanistic and pharmacological characterization of N-pyridazin-3-yl-4-(3-{[5-(trifluoromethyl)pyridin-2-yl]oxy}benzylidene)piperidine-1-carboxamide (PF-04457845), a highly efficacious and selective FAAH inhibitor. Mechanistic studies confirm that PF-04457845 is a time-dependent, covalent FAAH inhibitor that carbamylates FAAH's catalytic serine nucleophile. PF-04457845 inhibits human FAAH with high potency (k(inact)/K(i) = 40,300 M(-1)s(-1); IC(50) = 7.2 nM) and is exquisitely selective in vivo as determined by activity-based protein profiling. Oral administration of PF-04457845 produced potent antinociceptive effects in both inflammatory [complete Freund's adjuvant (CFA)] and noninflammatory (monosodium iodoacetate) pain models in rats, with a minimum effective dose of 0.1 mg/kg (CFA model). PF-04457845 displayed a long duration of action as a single oral administration at 1 mg/kg showed in vivo efficacy for 24 h with a concomitant near-complete inhibition of FAAH activity and maximal sustained elevation of anandamide in brain. Significantly, PF-04457845-treated mice at 10 mg/kg elicited no effect in motility, catalepsy, and body temperature. Based on its exceptional selectivity and in vivo efficacy, combined with long duration of action and optimal pharmacokinetic properties, PF-04457845 is a clinical candidate for the treatment of pain and other nervous system disorders.

Discovery of novel spirocyclic inhibitors of fatty acid amide hydrolase (FAAH). Part 2. Discovery of 7-azaspiro[3.5]nonane urea PF-04862853, an orally efficacious inhibitor of fatty acid amide hydrolase (FAAH) for pain.

Fatty acid amide hydrolase (FAAH) is an integral membrane serine hydrolase responsible for the degradation of fatty acid amide signaling molecules such as endocannabinoid anandamide (AEA), which has been shown to possess cannabinoid-like analgesic properties. Herein we report the optimization of spirocyclic 7-azaspiro[3.5]nonane and 1-oxa-8-azaspiro[4.5]decane urea covalent inhibitors of FAAH. Using an iterative design and optimization strategy, lead compounds were identified with a remarkable reduction in molecular weight and favorable CNS drug like properties. 3,4-Dimethylisoxazole and 1-methyltetrazole were identified as superior urea moieties for this inhibitor class. A dual purpose in vivo efficacy and pharmacokinetic screen was designed to be the key decision enabling experiment affording the ability to move quickly from compound synthesis to selection of preclinical candidates. On the basis of the remarkable potency, selectivity, pharmacokinetic properties and in vivo efficacy, PF-04862853 (15p) was advanced as a clinical candidate.

Discovery of novel spirocyclic inhibitors of fatty acid amide hydrolase (FAAH). Part 1: identification of 7-azaspiro[3.5]nonane and 1-oxa-8-azaspiro[4.5]decane as lead scaffolds.

Herein we report the identification of two new fatty acid amide hydrolase (FAAH) inhibitor lead series with FAAH k(inact)/K(i) potency values greater than 1500M(-1)s(-1). The two novel spirocyclic cores, 7-azaspiro[3.5]nonane and 1-oxa-8-azaspiro[4.5]decane, clearly distinguished themselves from the other spirocyclic cores on the basis of their superior potency for FAAH. Lead compounds from these two series have suitable FAAH potency and selectivity for additional medicinal chemistry optimization.

Proinflammatory cytokines, IL-1ß and TNF-α, induce expression of interleukin-34 mRNA via JNK- and p44/42 MAPK-NF-κB pathway but not p38 pathway in osteoblasts.

The aim of this study is to investigate the induction of interleukin-34 (IL-34) and macrophage colony-stimulating factor (M-CSF) mRNA by inflammatory cytokines and the involvement of mitogen-activated protein kinases (MAPKs) in this signaling pathway in human osteoblasts as both IL-34 and M-CSF bind to the same receptor c-FMS. Among four inflammatory cytokines [(IL-1ß, IL-6, IL-17, and tumor necrosis factor-α (TNF-α)], IL-34 mRNA expression level was dramatically induced by IL-1ß (17-fold) and TNF-α (74-fold). IL-1ß and TNF-α activated the intracellular mitogen-activated protein kinases (MAPKs): p44/42 MAPK, p38, and c-Jun N-terminal kinase (JNK) as well as nuclear factor-κB (NF-κB) in osteoblasts. IL-1ß- and TNF-α-mediated induction of IL-34 mRNA expression was decreased by JNK inhibitor. Interestingly, although treatment of MEK-1/2 inhibitor showed no reduction in the increase of IL-34 mRNA expression by cytokines, combination of MEK-1/2 inhibitor and JNK inhibitor significantly inhibited IL-1ß- and TNF-α-mediated IL-34 mRNA expression level compared to those by each inhibitor alone. On the other hand, M-CSF mRNA expression level was significantly induced by both IL-1ß and TNF-α by up to 7- and 11-fold, respectively. IL-1ß- and TNF-α-mediated induction of M-CSF mRNA was not affected by p38, JNK, and MEK-1/2 inhibitors. However, NF-κB inhibitor completely inhibited the elevation of M-CSF mRNA expression by these cytokines. These results showed that proinflammatory cytokines, IL-1ß and TNF-α, induced the expression of IL-34 mRNA via JNK and p44/42 MAPK but not p38 in human osteoblasts while p38, JNK, and p44/42 MAPK were not involved in the induction of M-CSF mRNA expression by these cytokines.

Macrophage-colony stimulating factor and interleukin-34 induce chemokines in human whole blood.

The aim of this study is to investigate if macrophage-colony stimulating factor (M-CSF) or interleukin-34 (IL-34) induces cytokines or chemokines using human whole blood (HWB) and if an M-CSF- or IL-34-induced cytokine or chemokine production from HWB is inhibited by soluble M-CSF receptor or c-FMS kinase inhibitors. Among eight cytokines or growth factors tested, only IL-6 level was increased by up to 6-fold by M-CSF or IL-34 in HWB. In contrast, chemokine levels (IP-10/CXCL10, IL-8/CXCL8, and MCP-1/CCL2) were dramatically increased by M-CSF or IL-34 in HWB while exhibiting a large variation among donors. Variability of the MCP-1 signal induced by M-CSF or IL-34 was relatively less among donors compared to the IP-10 and IL-8 signals. The elevation of these chemokine levels was significantly decreased by soluble M-CSF receptor, indicating the elevation of these chemokines was mediated by M-CSF or IL-34. Furthermore, GW2580, a c-FMS kinase inhibitor, inhibited the induction of MCP-1 by M-CSF or IL-34 in a concentration dependent manner. These indicate MCP-1 is the most appropriate chemokine target for a chemokine release assay to evaluate the potency of c-FMS kinase inhibitors and MCP-1 release assay using HWB would be useful, relevant tool for translational pharmacology of c-FMS kinase inhibitors.

Structure based design of novel irreversible FAAH inhibitors.

Fatty acid amide hydrolase (FAAH) has attracted significant attention due to its promise as an analgesic target. This has resulted in the discovery of numerous chemical classes as inhibitors of this potential therapeutic target. In this paper we disclose a new series of novel FAAH irreversible azetidine urea inhibitors. In general these compounds illustrate potent activity against the rat FAAH enzyme. Our SAR studies allowed us to optimize this series resulting in the identification of compounds 13 which were potent inhibitors of both human and rat enzyme. This series of compounds illustrated good hydrolase selectivity along with good PK properties.

Benzothiophene piperazine and piperidine urea inhibitors of fatty acid amide hydrolase (FAAH).

The synthesis and structure-activity relationships (SAR) of a series of benzothiophene piperazine and piperidine urea FAAH inhibitors is described. These compounds inhibit FAAH by covalently modifying the enzyme's active site serine nucleophile. Activity-based protein profiling (ABPP) revealed that these urea inhibitors were completely selective for FAAH relative to other mammalian serine hydrolases. Several compounds showed in vivo activity in a rat complete Freund's adjuvant (CFA) model of inflammatory pain.

PF-04447943, a Phosphodiesterase 9A Inhibitor, in Stable Sickle Cell Disease Patients: A Phase Ib Randomized, Placebo-Controlled Study.

This phase Ib study randomized patients with stable sickle cell disease (SCD) aged 18-65 years to twice-daily PF-04447943 (a phosphodiesterase 9A inhibitor; 5 or 25 mg) or placebo, with/without hydroxyurea coadministration, for up to 29 days. Blood samples were collected at baseline and various posttreatment time points for assessments of PF-04447943 pharmacokinetics (PKs)/pharmacodynamics (PDs). Change from baseline in potential SCD-related biomarkers was evaluated. Of 30 patients, 15 received hydroxyurea and 28 completed the study. PF-04447943, with/without hydroxyurea, was generally well tolerated, with no treatment-related serious adverse events. Plasma PF-04447943 exposure was dose proportional. Twice-daily PF-04447943 25 mg significantly reduced the number and size of circulating monocyte-platelet and neutrophil-platelet aggregates and levels of circulating soluble E-selectin at day 29 vs. baseline (adjusted P < 0.15). PF-04447943 demonstrated PK/PD effects suggestive of inhibiting pathways that may contribute to vaso-occlusion. This study also provides guidance regarding biomarkers for future SCD studies.

Discovery of PF-04457845: A Highly Potent, Orally Bioavailable, and Selective Urea FAAH Inhibitor.

Fatty acid amide hydrolase (FAAH) is an integral membrane serine hydrolase that degrades the fatty acid amide family of signaling lipids, including the endocannabinoid anandamide. Genetic or pharmacological inactivation of FAAH leads to analgesic and anti-inflammatory phenotypes in rodents without showing the undesirable side effects observed with direct cannabinoid receptor agonists, indicating that FAAH may represent an attractive therapeutic target for the treatment of inflammatory pain and other nervous system disorders. Herein, we report the discovery and characterization of a highly efficacious and selective FAAH inhibitor PF-04457845 (23). Compound 23 inhibits FAAH by a covalent, irreversible mechanism involving carbamylation of the active-site serine nucleophile of FAAH with high in vitro potency (k(inact)/K(i) and IC(50) values of 40300 M(-1) s(-1) and 7.2 nM, respectively, for human FAAH). Compound 23 has exquisite selectivity for FAAH relative to other members of the serine hydrolase superfamily as demonstrated by competitive activity-based protein profiling. Oral administration of 23 at 0.1 mg/kg results in efficacy comparable to that of naproxen at 10 mg/kg in a rat model of inflammatory pain. Compound 23 is being evaluated in human clinical trials.

Discovery and characterization of a highly selective FAAH inhibitor that reduces inflammatory pain.

Endocannabinoids are lipid signaling molecules that regulate a wide range of mammalian behaviors, including pain, inflammation, and cognitive/emotional state. The endocannabinoid anandamide is principally degraded by the integral membrane enzyme fatty acid amide hydrolase (FAAH), and there is currently much interest in developing FAAH inhibitors to augment endocannabinoid signaling in vivo. Here, we report the discovery and detailed characterization of a highly efficacious and selective FAAH inhibitor, PF-3845. Mechanistic and structural studies confirm that PF-3845 is a covalent inhibitor that carbamylates FAAH's serine nucleophile. PF-3845 selectively inhibits FAAH in vivo, as determined by activity-based protein profiling; raises brain anandamide levels for up to 24 hr; and produces significant cannabinoid receptor-dependent reductions in inflammatory pain. These data thus designate PF-3845 as a valuable pharmacological tool for in vivo characterization of the endocannabinoid system.