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The IQ Consortium NHP Reuse Working Group (WG) comprises members from 15 pharmaceutical and biotechnology companies. In 2020, the WG developed and distributed a detailed questionnaire on protein non-naïve NHP reuse to the WG member companies. The WG received responses from key stakeholders including principal investigators, facility managers, animal welfare officers and research scientists. This paper’s content reflects the consolidated opinion of the WG members and the questionnaire responses on the subject of NHP reuse within nonclinical programs at all stages of research and development. Many of the pharmaceutical companies represented in the working group or participating in the questionnaire have already achieved some level of NHP reuse in their nonclinical programs, but the survey results suggested that there is significant potential to increase NHP reuse further and a need to understand the considerations involved in reuse more clearly. The WG has also focused carefully on the inherent concerns and risks of implementing protein non-naive NHP reuse and has evaluated the best methods of risk assessment and decision-making. This paper presents a discussion on the challenges and opportunities surrounding protein non-naïve NHP reuse and aims to stimulate further industry dialogue on the subject and provide guidance for pharmaceutical companies to establish roadmaps and decision trees enabling increased protein non-naïve NHP reuse. In addition, this paper represents a solid basis for collaborative engagement between pharmaceutical and biotechnology companies with contract research organizations (CROs) to discuss how the availability of protein non-naïve NHP within CROs can be better leveraged for their use within nonclinical studies.

Structural elucidation of small molecules only available in low quantity (nanogram) is one of the big advantages of the crystalline sponge method. The optimization of various soaking parameters is crucial for effective analyte absorption and repetitive positioning in the pores of the crystal. Time-consuming X-ray diffraction measurements are necessary for data collection and confirmation of successful guest inclusion. In this work, we report a screening method to select optimal soaking conditions without the need of single-crystal X-ray diffraction analysis for individual compounds and mixtures. 14 substances were chosen as test compounds. Parallel guest soaking of individual compounds and mixtures was conducted using various soaking conditions. After evaporation of solvent, excessive material was removed, and guest molecules released through dissolution of the framework. Liquid chromatography-tandem mass spectrometry allowed the estimation of analyte trapped in the pores and the selection of optimal soaking condition dependent on the highest amount of analyte to crystal size (affinity factor). The tool allowed subsequent crystallographic analysis of ten compounds with minimal experiment time. Additionally, a study to examine the lower limit of detection of the crystalline sponge method was conducted. Determination of two target analytes was possible using only 5 ng of sample. Our study shows the potential of an affinity screening to prioritize soaking parameters by estimation of the guest concentration in a single crystal for one or multiple target compounds within a short period of time.

Herein, we report the discovery of a novel class of quinazoline carboxamides as dual p70S6k/Akt inhibitors for the treatment of tumors driven by alterations to the PI3K/Akt/mTOR (PAM) pathway. Through the screening of in-house proprietary kinase library, 4-benzylamino-quinazoline-8-carboxylic acid amide 1 stood out, with sub-micromolar p70S6k biochemical activity, as the starting point for a structurally enabled p70S6K/Akt dual inhibitor program that led to the discovery of M2698, a dual p70S6k/Akt inhibitor. M2698 is kinase selective, possesses favorable physical, chemical, and DMPK profiles, is orally available and well tolerated, and displayed tumor control in multiple in vivo studies of PAM pathway-driven tumors.

Pregnane X receptor (PXR), constitutive androstane receptor (CAR), and PXR/CAR knockout (KO) HepaRG cells, as well as a PXR reporter gene assay, were used to investigate the mechanism of CYP3A4 and CYP2B6 induction by prototypical substrates and a group of compounds from the Merck KGaA oncology drug discovery pipeline. The basal and inducible gene expression of CYP3A4 and CYP2B6 of nuclear hormone receptor (NHR) KO HepaRG relative to control HepaRG was characterized. The basal expression of CYP3A4 was markedly higher in the PXR (10-fold) and CAR (11-fold) KO cell lines compared with control HepaRG, whereas inducibility was substantially lower. Inversely, basal expression of CYP3A4 in PXR/CAR double KO (dKO) was low (10-fold reduction). Basal CYP2B6 expression was high in PXR KO (9-fold) cells which showed low inducibility, whereas the basal expression remained unchanged in CAR and dKO cell lines compared with control cells. Most of the test compounds induced CYP3A4 and CYP2B6 via PXR and, to a lesser extent, via CAR. Furthermore, other non-NHR-driven induction mechanisms were implicated, either alone or in addition to NHRs. Notably, 5 of the 16 compounds (31%) that were PXR inducers in HepaRG did not activate PXR in the reporter gene assay, illustrating the limitations of this system. This study indicates that HepaRG is a highly sensitive system fit for early screening of cytochrome P450 (P450) induction in drug discovery. Furthermore, it shows the applicability of HepaRG NHR KO cells as tools to deconvolute mechanisms of P450 induction using novel compounds representative for oncology drug discovery. SIGNIFICANCE STATEMENT: This work describes the identification of induction mechanisms of CYP3A4 and CYP2B6 for an assembly of oncology drug candidates using HepaRG nuclear hormone receptor knockout and displays its advantages compared to a pregnane X receptor reporter gene assay. With this study, risk assessment of drug candidates in early drug development can be improved.

The human absorption, distribution, metabolism, and excretion (hADME) study is the cornerstone of the clinical pharmacology package for small molecule drugs, providing comprehensive information on the rates and routes of disposition and elimination of drug-related material in humans through the use of

Accurate prediction of human pharmacokinetics using in vitro tools is an important task during drug development. Albeit, currently used in vitro systems for clearance extrapolation such as microsomes and primary human hepatocytes in suspension culture show reproducible turnover, the utility of these systems is limited by a rapid decline of activity of drug metabolizing enzymes. In this study, a multi-well array culture of primary human hepatocyte spheroids was compared to suspension and single spheroid cultures from the same donor. Multi-well spheroids remained viable and functional over the incubation time of 3 days, showing physiological excretion of albumin and α-AGP. Their metabolic activity was similar compared to suspension and single spheroid cultures. This physiological activity, the high cell concentration, and the prolonged incubation time resulted in significant turnover of all tested low clearance compounds (n = 8). In stark contrast, only one or none of the compounds showed significant turnover when single spheroid or suspension cultures were used. Using multi-well spheroids and a regression offset approach (log(CL

Praziquantel (PZQ) is the drug of choice for treatment of the neglected tropical disease schistosomiasis. Although the drug has been extensively used over several decades and its metabolism well studied (several oxidative metabolites are known from literature), the knowledge of the complete structure of some of its metabolites remains elusive. Conventional techniques, such as nuclear magnetic resonance or liquid chromatography mass spectrometry were used in the past to investigate phase I and phase II metabolites of PZQ. These techniques are either limited to provide the complete molecular structure (liquid chromatography mass spectrometry) or require large amount of sample material (NMR), which are not always available when

Understanding the metabolism of new drug candidates is important during drug discovery and development, as circulating metabolites may contribute to efficacy or cause safety issues. In the early phase of drug discovery, human in vitro systems are used to investigate human relevant metabolism. Though conventional techniques are limited in their ability to provide complete molecular structures of metabolites (liquid chromatography mass spectrometry) or require a larger amount of material not available from in vitro incubation (nuclear magnetic resonance), we here report for the first time the use of the crystalline sponge method to identify phase I and phase II metabolites generated from in vitro liver microsomes or S9 fractions. Gemfibrozil was used as a test compound. Metabolites generated from incubation with microsomes or S9 fractions, were fractionated using online fraction collection. After chromatographic purification and fractionation of the generated metabolites, single crystal X-ray diffraction of crystalline sponges was used to identify the structure of gemfibrozil metabolites. This technique allowed for complete structure elucidation of 5'-CH2OH gemfibrozil (M1), 4'-OH gemfibrozil (M2), 5'-COOH gemfibrozil (M3), and the acyl glucuronide of gemfibrozil, 1-O-β-glucuronide (M4), the first acyl glucuronide available in the Cambridge Crystallographic Data Centre. Our study shows that when optimal soaking is possible, crystalline sponges technology is a sensitive (nanogram amount) and fast (few days) method that can be applied early in drug discovery to identify the structure of pure metabolites from in vitro incubations. SIGNIFICANCE STATEMENT: Complete structure elucidation of human metabolites plays a critical role in early drug discovery. Low amounts of material (nanogram) are only available at this stage and insufficient for nuclear magnetic resonance analysis. The crystalline sponge method has the potential to close this gap, as demonstrated in this study.

Immunoglobulin A nephropathy (IgAN) is a leading cause of chronic kidney disease, frequently associated with hypertension and renal inflammation. ω-3 fatty acids eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) in fish oil (FO) improve kidney function in animal models, but have inconsistent metabolic effects in humans. Oxylipin profiles in serum from IgAN patients supplemented with either FO or corn oil (CO) placebo were analyzed by liquid chromatography coupled to tandem mass spectrometry. EPA cyclooxygenase and lipoxygenase metabolites, and EPA and DHA epoxides and diols were increased in response to FO supplementation, as were total epoxides and epoxide/diol ratios. Several of these metabolites were drivers of separation as assessed by multivariate analysis of FO patients pre- vs. post-supplementation, including 17,18-dihydroxyeicosatrienoic acid, prostaglandin D3, prostagalandin E3, Resolvin E1, 12-hydroxyeicosapentaenoic acid, and 10(11)-epoxydocosapentaenoic acid. In patients whose proteinuria improved, plasma total oxylipins as well as several hydroxyoctadecadienoic acids, hydroxyeicosatetraenoic acids, and leukotriene B4 metabolites were among the metabolites that were significantly lower than in patients whose proteinuria either did not improve or worsened. These data support the involvement of oxylipins in the inflammatory component of IgAN as well as the potential use of oxylipin profiles as biomarkers and for assessing responsiveness to ω-3 fatty acid supplementation in IgAN patients.

Cyclooxygenase, lipoxygenase, and epoxygenase derived oxylipins, especially eicosanoids, play important roles in many physiological processes. Assessment of oxidized fatty acid levels is important for understanding their homeostatic and pathophysiological roles. Most reported methods examine these pathways in isolation. The work described here employed a solid phase extraction-liquid chromatography-electrospray ionization MS/MS (SPE-LC-ESI MS/MS) method to monitor these metabolites. In 21 min, 39 oxylipins were quantified along with eight corresponding internal standards. The limits of quantification were between 0.07 and 32 pg (20 pM-10 nM). Finally, the validated method was used to evaluate oxylipin profiles in lipopolysaccharide-exposed mice, an established septic inflammatory model. The method described here offers a useful tool for the evaluation of complex regulatory oxylipin responses in in vitro or in vivo studies.

Soluble epoxide hydrolase (sEH) metabolizes epoxyeicosatrienoic acids and represents a novel therapeutic target in cardiovascular disease treatment. We investigated the relationship among sequence variation in the sEH gene (Ephx2), sEH function, and risk of end-organ injury in strains of spontaneously hypertensive rat (SHRs) differing in their susceptibility to develop brain vascular disease. Brain Ephx2 expression was significantly lower in stroke-prone (SHR/A3) than in stroke-resistant (SHR/N) SHRs (5-fold; P P P

Introduction There is a high degree of inter-individual variability among people in response to intervention with omega-3 fatty acids (FA), which may partly explain conflicting results on the effectiveness of omega-3 FA for the treatment and prevention of chronic inflammatory diseases. In this study we sought to evaluate whether part of this inter-individual variability in response is related to the regulation of key oxylipin metabolic genes in circulating peripheral blood mononuclear cells (PBMCs). Methods Plasma FA and oxylipin profiles from 12 healthy individuals were compared to PBMC gene expression profiles following six weeks of supplementation with fish oil, which delivered 1.9 g/d eicosapentaenoic acid (EPA) and 1.5 g/d docosahexaenoic acid (DHA). Fold changes in gene expression were measured by a quantitative polymerase chain reaction (qPCR). Results Healthy individuals supplemented with omega-3 FA had differential responses in prostaglandin-endoperoxide synthase 1 (PTGS1), prostaglandin-endoperoxide synthase 2 (PTGS2), arachidonate 12-lipoxygenase (ALOX12), and interleukin 8 (IL-8) gene expression in isolated PBMCs. In those individuals for whom plasma arachidonic acid (ARA) in the phosphatidylethanolamine (PE) lipid class decreased in response to omega-3 intervention, there was a corresponding decrease in gene expression for PTGS1 and ALOX12. Several oxylipin product/FA precursor ratios (e.g. prostaglandin E2 (PGE2)/ARA for PTGS1 and 12-hydroxyeicosatetraenoic acid (12-HETE)/ARA for ALOX12) were also associated with fold change in gene expression, suggesting an association between enzyme activity and gene expression. The fold-change in PTGS1 gene expression was highly positively correlated with ALOX12 gene expression but not with PTGS2, whereas IL-8 and PTGS2 were positively correlated. Conclusions The regulation of important oxylipin metabolic genes in PBMCs varied with the extent of change in ARA concentrations in the case of PTGS1 and ALOX12 regulation. PBMC gene expression changes in response to omega-3 supplementation varied among healthy individuals, and were associated with changes in plasma FA and oxylipin composition to different degrees in different individuals. Trial Registration clinicaltrials.gov NCT01838239

Sphingosine-1-phosphate (S1P) receptor agonists have shown promise as therapeutic agents for multiple sclerosis (MS) due to their regulatory roles within the immune, central nervous system, and cardiovascular system. Here, the design and optimization of novel [1,2,4]oxadiazole derivatives as selective S1P receptor agonists are described. The structure-activity relationship exploration was carried out on the three dominant segments of the series: modification of the polar head group (P), replacement of the oxadiazole linker (L) with different five-membered heterocycles, and the use of diverse 2,2'-disubstituted biphenyl moieties as the hydrophobic tail (H). All three segments have a significant impact on potency, S1P receptor subtype selectivity, physicochemical properties, and in vitro absorption, distribution, metabolism, excretion and toxicity (ADMET) profile of the compounds. From these optimization studies, a selective S1P1 agonist, N-methyl-N-(4-{5-[2-methyl-2'-(trifluoromethyl)biphenyl-4-yl]-1,2,4-oxadiazol-3-yl}benzyl)glycine (45), and a dual S1P1,5 agonist, N-methyl-N-(3-{5-[2'-methyl-2-(trifluoromethyl)biphenyl-4-yl]-1,2,4-oxadiazol-3-yl}benzyl)glycine (49), emerged as frontrunners. These compounds distribute predominantly in lymph nodes and brain over plasma and induce long lasting decreases in lymphocyte count after oral administration. When evaluated head-to-head in an experimental autoimmune encephalomyelitis mouse model, together with the marketed drug fingolimod, a pan-S1P receptor agonist, S1P1,5 agonist 49 demonstrated comparable efficacy while S1P1 -selective agonist 45 was less potent. Compound 49 is not a prodrug, and its improved property profile should translate into a safer treatment of relapsing forms of MS.

During inflammation, a large amount of arachidonic acid (AA) is released into the cellular milieu and cyclooxygenase enzymes convert this AA to prostaglandins that in turn sensitize pain pathways. However, AA is also converted to natural epoxyeicosatrienoic acids (EETs) by cytochrome P450 enzymes. EET levels are typically regulated by soluble epoxide hydrolase (sEH), the major enzyme degrading EETs. Here we demonstrate that EETs or inhibition of sEH lead to antihyperalgesia by at least 2 spinal mechanisms, first by repressing the induction of the COX2 gene and second by rapidly up-regulating an acute neurosteroid-producing gene, StARD1 , which requires the synchronized presence of elevated cAMP and EET levels. The analgesic activities of neurosteroids are well known; however, here we describe a clear course toward augmenting the levels of these molecules. Redirecting the flow of pronociceptive intracellular cAMP toward up-regulation of StARD1 mRNA by concomitantly elevating EETs is a novel path to accomplish pain relief in both inflammatory and neuropathic pain states.

Introduction: Conflicting findings in both interventional and observational studies have resulted in a lack of consensus on the benefits of omega 3 fatty acids in reducing disease risk. This may be due to individual variability in response. We used a multi-platform lipidomic approach to investigate both the consistent and inconsistent responses of individuals comprehensively to a defined omega 3 intervention. Methods: The lipidomic profile including fatty acids, lipid classes, lipoprotein distribution, and oxylipins was examined multi- and uni-variately in 12 healthy subjects pre vs. post six weeks of omega 3 fatty acids (1.9 g/d eicosapentaenoic acid [EPA] and 1.5 g/d docosahexaenoic acid [DHA]). Results: Total lipidomic and oxylipin profiles were significantly different pre vs. post treatment across all subjects (p=0.00007 and p=0.00002 respectively). There was a strong correlation between oxylipin profiles and EPA and DHA incorporated into different lipid classes (r(2)=0.93). However, strikingly divergent responses among individuals were also observed. Both omega 3 and omega 6 fatty acid metabolites displayed a large degree of variation among the subjects. For example, in half of the subjects, two arachidonic acid cyclooxygenase products, prostaglandin E-2 (PGE(2)) and thromboxane B2 (TXB2), and a lipoxygenase product, 12-hydroxyeicosatetraenoic acid (12-HETE) significantly decreased post intervention, whereas in the other half they either did not change or increased. The EPA lipoxygenase metabolite 12-hydroxyeicosapentaenoic acid (12-HEPE) varied among subjects from an 82% decrease to a 5,000% increase. Conclusions: Our results show that certain defined responses to omega 3 fatty acid intervention were consistent across all subjects. However, there was also a high degree of inter-individual variability in certain aspects of lipid metabolism. This lipidomic based phenotyping approach demonstrated that individual responsiveness to omega 3 fatty acids is highly variable and measurable, and could be used as a means to assess the effectiveness of omega 3 interventions in modifying disease risk and determining metabolic phenotype.