Qualification of translational safety biomarkers.

Safety biomarkers are important drug development tools, both preclinically and clinically. It is a straightforward process to correlate the performance of nonclinical safety biomarkers with histopathology, and ideally, the biomarker is useful in all species commonly used in safety assessment. In clinical validation studies, where histopathology is not feasible, safety biomarkers are compared to the response of standard biomarkers and/or to clinical adjudication. Worldwide, regulatory agencies have put in place processes to qualify biomarkers to provide confidence in the manner of use and interpretation of biomarker data in drug development studies. This paper describes currently qualified safety biomarkers which can be utilized to monitor for nephrotoxicity and cardiotoxicity and ongoing projects to qualify safety biomarkers for liver, skeletal muscle, and vascular injury. In many cases, the development and use of these critical drug development tools is dependent upon partnerships and the precompetitive sharing of data to support qualification efforts.

Biomarkers of Crohn's Disease to Support the Development of New Therapeutic Interventions.

BACKGROUND: Currently, 2 coprimary end points are used by health authorities to determine the effectiveness of therapeutic interventions in patients with Crohn's disease (CD): symptomatic remission (patient-reported outcome assessment) and endoscopic remission (ileocolonoscopy). However, there is lack of accepted biomarkers to facilitate regulatory decision-making in the development of novel therapeutics for the treatment of CD. METHODS: With support from the Helmsley Charitable Trust, Critical Path Institute formed the Crohn's Disease Biomarkers preconsortium (CDBpC) with members from the pharmaceutical industry, academia, and nonprofit organizations to evaluate the CD biomarker landscape. Biomarkers were evaluated based on biological relevance, availability of biomarker assays, and clinical validation data. RESULTS: The CDBpC identified the most critical need as pharmacodynamic/response biomarkers to monitor disease activity in response to therapeutic intervention. Fecal calprotectin (FC) and serum C-reactive protein (CRP) were identified as biomarkers ready for the regulatory qualification process. A number of exploratory biomarkers and potential panels of these biomarkers was also identified for additional development. Given the different factors involved in CD and disease progression, a combination of biomarkers, including inflammatory, tissue injury, genetic, and microbiome-associated biomarkers, will likely have the most utility. CONCLUSIONS: The primary focus of the Inflammatory Bowel Disease Regulatory Science Consortium will be development of exploratory biomarkers and the qualification of FC and CRP for IBD. The Inflammatory Bowel Disease Regulatory Science Consortium, focused on tools to support IBD drug development, will operate in the precompetitive space to share data, biological samples for biomarker testing, and assay information for novel biomarkers.

Serum glutamate dehydrogenase activity enables early detection of liver injury in subjects with underlying muscle impairments.

Serum activities of alanine and aspartate aminotransferases (ALT and AST) are used as gold standard biomarkers for the diagnosis of hepatocellular injury. Since ALT and AST lack liver specificity, the diagnosis of the onset of hepatocellular injury in patients with underlying muscle impairments is severely limited. Thus, we evaluated the potential of glutamate dehydrogenase (GLDH) as a liver specific alternative biomarker of hepatocellular injury. In our study, serum GLDH in subjects with Duchene muscular dystrophy (DMD) was equivalent to serum GLDH in age matched healthy subjects, while serum ALT was increased 20-fold in DMD subjects. Furthermore, serum GLDH in 131 subjects with variety of muscle impairments was similar to serum GLDH of healthy subjects while serum ALT corelated with serum creatine kinase, a widely accepted biomarker of muscle impairment. In addition, significant elevations of ALT, AST, and CK were observed in a case of a patient with rhabdomyolysis, while serum GLDH stayed within the normal range until the onset of hypoxia-induced liver injury. In a mouse model of DMD (DMDmdx), serum GLDH but not serum ALT clearly correlated with the degree of acetaminophen-induced liver injury. Taken together, our data support the utility of serum GLDH as a liver-specific biomarker of liver injury that has a potential to improve diagnosis of hepatocellular injury in patients with underlying muscle impairments. In drug development, GLDH may have utility as a biomarker of drug induced liver injury in clinical trials of new therapies to treat muscle diseases such as DMD.

The importance of drug safety and tolerability in the development of new immunosuppressive therapy for transplant recipients: The Transplant Therapeutics Consortium's position statement.

The Transplant Therapeutics Consortium (TTC) is a public-private partnership between the US Food and Drug Administration and the transplantation community including the transplantation societies and members of the biopharmaceutical industry. The TTC was formed to accelerate the process of developing new medical products for transplant patients. The initial goals of this collaboration are the following: (a) To define which aspects of the kidney transplant drug-development process have clear needs for improvement from an industry and regulatory perspective; (b) to define which of the unmet needs in the process could be positively impacted through the development of specific drug-development tools based on available data; and (c) to determine the most appropriate pathway to achieve regulatory acceptance of the proposed process-accelerating tools. The TTC has identified 2 major areas of emphasis: new biomarkers or endpoints for determining the efficacy of new therapies and new tools to assess the safety or tolerability of new therapies. This article presents the rationale and planned approach to develop new tools to assess safety and tolerability of therapies for transplant patients. We also discuss how similar efforts might support the continued development of patient-reported outcome measures in the future.

Preclinical biomarker qualification.

Biomarkers are ubiquitously used within drug development programs in both nonclinical species and in humans to assess safety and efficacy of novel compounds. To routinely apply such novel biomarkers with certainty, a well-defined data package is necessary for review and endorsement by regulatory agencies including the US Food and Drug Administration, European Medicines Agency, and Japanese Pharmaceuticals and Medical Devices Agency. This type of endorsement is known as regulatory qualification. Novel approaches are being applied to speed the process, lower the resource intensity, and increase the accessibility of biomarker qualification data and it is likely that consortia will continue to play a fundamental role in the qualification process by bringing together like-minded stakeholders focused on specific tools to accelerate drug development. This article will focus on learnings from the previous three nonclinical biomarker qualification projects, as well as discuss the progression of preclinical biomarker projects into the clinical qualification space and the current strategy for the use of nonclinical biomarker data in the translational qualification of clinical biomarkers; much like nonclinical information is used in the approval of drug development candidates. Impact statement This minireview provides an overview of the history of preclinical biomarker qualification by summarizing the three examples of this type of qualification with US Food and Drug Administration, European Medicines Agency, and Japanese Pharmaceuticals and Medical Devices Agency. In addition, an overview of the biomarker qualification process is included to educate key stakeholders with links to relevant white papers that provide information on current evidentiary considerations. The manuscript also provides new information on the evolution of the role that preclinical qualification plays in clinical qualification of biomarkers and the novel approaches that are being utilized to improve the process.

The 5th Annual One Mind Summit: Lessons Learned About "Science Informing Brain Health Policies and Practice".

Advances in science frequently precede changes in clinical care by several years or even decades. To better understand the path to translation, we invited experts to share their perspectives at the 5th Annual One Mind Summit: "Science Informing Brain Health Policies and Practice," which was held on May 24-25, 2016, in Crystal City, VA. While the translation of brain research throughout the pipeline-from basic science research to patient care-was discussed, the focus was on the implementation of "best evidence" into patient care. The Summit identified key steps, including the need for professional endorsement and clinical guidelines or policies, acceptance by regulators and payers, dissemination and training for clinicians, patient advocacy, and learning healthcare models. The path to implementation was discussed broadly, as well as in the context of a specific project to implement concussion screening in emergency and urgent care centers throughout the United States.

Discovery and molecular basis of potent noncovalent inhibitors of fatty acid amide hydrolase (FAAH).

Fatty acid amide hydrolase (FAAH), an amidase-signature family member, is an integral membrane enzyme that degrades lipid amides including the endogenous cannabinoid anandamide and the sleep-inducing molecule oleamide. Both genetic knock out and pharmacological administration of FAAH inhibitors in rodent models result in analgesic, anxiolytic, and antiinflammatory phenotypes. Targeting FAAH activity, therefore, presents a promising new therapeutic strategy for the treatment of pain and other neurological-related or inflammatory disorders. Nearly all FAAH inhibitors known to date attain their binding potency through a reversible or irreversible covalent modification of the nucleophile Ser241 in the unusual Ser-Ser-Lys catalytic triad. Here, we report the discovery and mechanism of action of a series of ketobenzimidazoles as unique and potent noncovalent FAAH inhibitors. Compound 2, a representative of these ketobenzimidazoles, was designed from a series of ureas that were identified from high-throughput screening. While urea compound 1 is characterized as an irreversible covalent inhibitor, the cocrystal structure of FAAH complexed with compound 2 reveals that these ketobenzimidazoles, though containing a carbonyl moiety, do not covalently modify Ser241. These inhibitors achieve potent inhibition of FAAH activity primarily from shape complementarity to the active site and through numerous hydrophobic interactions. These noncovalent compounds exhibit excellent selectivity and good pharmacokinetic properties. The discovery of this distinctive class of inhibitors opens a new avenue for modulating FAAH activity through nonmechanism-based inhibition.

Identification of potent, noncovalent fatty acid amide hydrolase (FAAH) inhibitors.

Starting from a series of ureas that were determined to be mechanism-based inhibitors of FAAH, several spirocyclic ureas and lactams were designed and synthesized. These efforts identified a series of novel, noncovalent FAAH inhibitors with in vitro potency comparable to known covalent FAAH inhibitors. The mechanism of action for these compounds was determined through a combination of SAR and co-crystallography with rat FAAH.

Rapid high-energy microwave fixation is required to determine the anandamide (N-arachidonoylethanolamine) concentration of rat brain.

Anandamide (N-arachidonoylethanolamine, AEA) is the putative endogenous ligand for the CB1 receptor. Despite being regulated enzymatically, brain AEA concentrations are quite variable and have been reported to increase in response to ischemia and post-mortem delay. Because these observations are similar to the effects of decapitation on brain concentrations of unesterified arachidonic acid and several of its metabolites, we propose that brain AEA concentrations also increase with decapitation and that immediate head-focused microwave irradiation is necessary to quantify basal brain AEA levels correctly. To test this hypothesis, we measured brain AEA levels in rats that were subjected to head-focused microwave irradiation 5 min. following decapitation (5.5 kW, 3.4 s) (ischemic) and prior to decapitation (controls). Brain AEA concentrations were quantified by LC/MS/MS. AEA concentrations from ischemic animals (10.01 +/- 4.41 pmol/g, mean +/- SD) were significantly higher and more variable than control concentrations (2.45 +/- 0.39 pmol/g). Thus, the basal concentration of AEA in the brain is lower than previously thought and future studies attempting to quantify brain AEA should consider using head-focused microwave fixation to prevent anomalous results.

A role for caveolae/lipid rafts in the uptake and recycling of the endogenous cannabinoid anandamide.

The mechanisms responsible for the uptake and cellular processing of the endogenous cannabinoid anandamide are not well understood. We propose that anandamide uptake may occur via a caveola/lipid raft-related endocytic process in RBL-2H3 cells. Inhibitors of caveola-related (clathrin-independent) endocytosis reduced anandamide transport by approximately 50% compared with the control. Fluorescein derived from fluorescently labeled anandamide colocalized with protein markers of caveolae at early time points following transport. In this study, we have also identified a yet unrecognized process involved in trafficking events affecting anandamide following its uptake. Following uptake of [(3)H]anandamide by RBL-2H3 cells, we found an accumulation of tritium in the caveolin-rich membranes. Inhibitors of both anandamide uptake and metabolism blocked the observed enrichment of tritium in the caveolin-rich membranes. Mass spectrometry of subcellular membrane fractions revealed that the tritium accumulation observed in the caveolin-rich membrane fraction was not representative of intact anandamide, suggesting that following metabolism by the enzyme fatty acid amide hydrolase (FAAH), anandamide metabolites are rapidly enriched in caveolae. Furthermore, HeLa cells, which do not express high levels of FAAH, showed an accumulation of tritium in the caveolin-rich membrane fraction only when transfected with FAAH cDNA. Western blot and immunocytochemistry analyses of RBL-2H3 cells revealed that FAAH was localized in intracellular compartments distinct from caveolin-1 localization. Together, these data suggest that following uptake via caveola/lipid raft-related endocytosis, anandamide is rapidly metabolized by FAAH, with the metabolites efficiently recycled to caveolin-rich membrane domains.

Alpha-2 adrenergic receptors stimulate actin organization in developing fetal rat cardiac myocytes.

Expression of alpha(2)-adrenergic receptors (alpha(2)-AR) is very high in fetal rat heart although numbers decline with increasing gestational age. The current experiments were designed to identify the subtypes of alpha(2)-AR expressed and the function of these receptors in fetal cardiac myocytes. Expression of alpha(2)A and alpha(2)C, but not alpha(2)B, was confirmed in the myocyte population by indirect immunofluorescence microscopy with subtype-specific antibodies and by Western blot. Both dexmedetomidine, an alpha(2)-selective agonist, and norepinephrine, increased actin cytoskeleton organization and this increase was blocked by the alpha(2)-selective antagonist, atipamezole. Furthermore, dexmedetomidine inhibited isoproterenol-stimulated cAMP accumulation in isolated fetal rat heart and this was blocked by rauwolscine. Therefore, functional alpha(2)A and alpha(2)B subtypes are present in the fetal rat heart where they may have a role in cardiac development.

M1 muscarinic receptor signaling in mouse hippocampus and cortex.

The five subtypes (M1-M5) of muscarinic acetylcholine receptors signal through G(alpha)(q) or G(alpha)(i)/G(alpha)(o). M1, M3 and M5 receptors couple through G(alpha)(q) and function predominantly as postsynaptic receptors in the central nervous system. M1 and M3 receptors are localized to brain regions involved in cognition, such as hippocampus and cortex, but their relative contribution to function has been difficult to ascertain due to the lack of subtype specific ligands. A functional and genetic approach was used to identify the predominant muscarinic receptor subtype(s) mediating responses in mouse hippocampus and cortex, as well as the relative degree of spare muscarinic receptors in hippocampus. The nonselective muscarinic agonist oxotremorine-M stimulated G(alpha)(q)/11-specific GTP-gamma-35S binding in a concentration dependent manner with a Hill slope near unity in wild type mouse hippocampus and cortex. Muscarinic receptor stimulated G(alpha)(q)/11-specific GTP-gamma-35S binding was virtually abolished in both the hippocampus and cortex of M1 receptor knockout (KO) mice. In contrast, there was no loss of signaling in M3 receptor KO mice in either brain region. Muscarinic receptor reserve in wildtype mouse hippocampus was measured by Furchgott analysis after partial receptor alkylation with propylbenzylcholine mustard. Occupation of just 15% of the M1 receptors in mouse hippocampus was required for maximal efficacy of oxotremorine-M-stimulated GTP-gamma-35S binding indicating a substantial level of spare receptors. These findings support a role for the M1 receptor subtype as the primary G(alpha)(q)/11-coupled muscarinic receptor in mouse hippocampus and cortex.

Characterization of a novel endocannabinoid, virodhamine, with antagonist activity at the CB1 receptor.

The first endocannabinoid, anandamide, was discovered in 1992. Since then, two other endocannabinoid agonists have been identified, 2-arachidonyl glycerol and, more recently, noladin ether. Here, we report the identification and pharmacological characterization of a novel endocannabinoid, virodhamine, with antagonist properties at the CB1 cannabinoid receptor. Virodhamine is arachidonic acid and ethanolamine joined by an ester linkage. Concentrations of virodhamine measured by liquid chromatography atmospheric pressure chemical ionization-tandem mass spectrometry in rat brain and human hippocampus were similar to anandamide. In peripheral tissues that express the CB2 cannabinoid receptor, virodhamine concentrations were 2- to 9-fold higher than anandamide. In contrast to previously described endocannabinoids, virodhamine was a partial agonist with in vivo antagonist activity at the CB1 receptor. However, at the CB2 receptor, virodhamine acted as a full agonist. Transport of [(14)C]anandamide by RBL-2H3 cells was inhibited by virodhamine. Virodhamine produced hypothermia in the mouse and acted as an antagonist in the presence of anandamide both in vivo and in vitro. As a potential endogenous antagonist at the CB1 receptor, virodhamine adds a new form of regulation to the endocannabinoid system.