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A clear scientific and operational need exists for harmonized bioanalytical immunogenicity study reporting to facilitate communication of immunogenicity findings and expedient review by industry and health authorities. To address these key bioanalytical reporting gaps and provide a report structure for documenting immunogenicity results, this cross-industry group was formed to establish harmonized recommendations and a develop a submission template to facilitate agency filings. Provided here are recommendations for reporting clinical anti-drug antibody (ADA) assay results using ligand-binding assay technologies. This publication describes the essential bioanalytical report (BAR) elements such as the method, critical reagents and equipment, study samples, results, and data analysis, and provides a template for a suggested structure for the ADA BAR. This publication focuses on the content and presentation of the bioanalytical ADA sample analysis report. The interpretation of immunogenicity data, including the evaluation of the impact of ADA on safety, exposure, and efficacy, is out of scope of this publication. Graphical Abstract

Evolving immunogenicity assay performance expectations and a lack of harmonized anti-drug antibody validation testing and reporting tools have resulted in significant time spent by health authorities and sponsors on resolving filing queries. Following debate at the American Association of Pharmaceutical Sciences National Biotechnology Conference, a group was formed to address these gaps. Over the last 3 years, 44 members from 29 organizations (including 5 members from Europe and 10 members from FDA) discussed gaps in understanding immunogenicity assay requirements and have developed harmonization tools for use by industry scientists to facilitate filings to health authorities. Herein, this team provides testing and reporting strategies and tools for the following assessments: (1) pre-study validation cut point; (2) in-study cut points, including procedures for applying cut points to mixed populations; (3) system suitability control criteria for in-study plate acceptance; (4) assay sensitivity, including the selection of an appropriate low positive control; (5) specificity, including drug and target tolerance; (6) sample stability that reflects sample storage and handling conditions; (7) assay selectivity to matrix components, including hemolytic, lipemic, and disease state matrices; (8) domain specificity for multi-domain therapeutics; (9) and minimum required dilution and extraction-based sample processing for titer reporting. Graphical Abstract

The SAR study of a series of 6-aryloxymethyl-8-aryl substituted quinolines is described. Optimization of the series led to the discovery of compound 26b, a highly potent (IC50=0.6 nM) and selective PDE4D inhibitor with a 75-fold selectivity over the A, B, and C subtypes and over 18,000-fold selectivity against other PDE family members. Rat pharmacokinetics and tissue distribution are also summarized.

The discovery and SAR of a new series of substituted 8-arylquinoline PDE4 inhibitors are herein described. This work has led to the identification of several compounds with excellent in vitro and in vivo profiles, including a good therapeutic window of emesis to efficacy in several animal models. Typical optimized compounds from this series are potent inhibitors of PDE4 (IC(50)

L-826,141 [4-(2-(3,4-bis-difluromethoxyphenyl)-2-(4-(1,1,1, 3,3,3-hexafluoro-2-hydroxypropan-2-yl)-phenyl]-ethyl)-3-methylpyridine-1-oxide] is a selective and potent inhibitor of phosphodiesterase 4 (PDE4) with an IC(50) value of 0.26 to 2.4 nM for inhibition of the catalytic activity of PDE4A, B, C, and D. The cAMP elevation that can be maintained by PDE4 inhibitors attenuates the signaling cascades that lead to the production of certain cytokines. In cellular-based assays, L-826,141 transcriptionally down-regulates production of tumor necrosis factor (TNF)-alpha in peripheral blood mononuclear cell and whole blood assays with IC(50) values of 31 and 310 nM, respectively. Profiling the effect of this compound on various cytokines in the signaling cascade attenuated by cAMP elevation demonstrates that L-826,141 is also a potent inhibitor of interleukin (IL)-12, granulocyte macrophage-colony stimulating factor, and interferon (IFN)gamma (IC(50) values of 0.3-0.9 microM) as well as TNF-alpha formation. We have also shown that the PDE4 inhibitors rolipram and L-826,141 are potent inhibitors of CD3-plus CD28-stimulated IL-2 production in naive human T cells. To address the effect of PDE4 inhibitors on cytokine release from T helper (Th)1 and Th2 effector cells, we used a well characterized model in which T cells are derived from ovalbumin (323-339)-specific T cell receptor transgenic mice. L-826,141 inhibits Th0-mediated IL-2 production with an IC(35) value of 25 nM and Th1-mediated IFNgamma production with an IC(30) value of 46 nM. In contrast, L-826,141 had no significant inhibitory effect (IC(30) value > 2.5 microM) on Th2 cell-mediated IL-4 nor IL-13 production. Together, these data demonstrate that specific inhibition of PDE4 preferentially blocks the production of Th1 versus Th2 effector cytokines in vitro.

Vanadate and pervanadate (the complexes of vanadate with hydrogen peroxide) are two commonly used general protein-tyrosine phosphatase (PTP) inhibitors. These compounds also have insulin-mimetic properties, an observation that has generated a great deal of interest and study. Since a careful kinetic study of the two inhibitors has been lacking, we sought to analyze their mechanisms of inhibition. Our results show that vanadate is a competitive inhibitor for the protein-tyrosine phosphatase PTP1B, with a Ki of 0.38+/-0.02 microM. EDTA, which is known to chelate vanadate, causes an immediate and complete reversal of the inhibition due to vanadate when added to an enzyme assay. Pervanadate, by contrast, inhibits by irreversibly oxidizing the catalytic cysteine of PTP1B, as determined by mass spectrometry. Reducing agents such as dithiothreitol that are used in PTP assays to keep the catalytic cysteine reduced and active were found to convert pervanadate rapidly to vanadate. Under certain conditions, slow time-dependent inactivation by vanadate was observed; since catalase blocked this inactivation, it was ascribed to in situ generation of hydrogen peroxide and subsequent formation of pervanadate. Implications for the use of these compounds as inhibitors and rationalization for some of their in vivo effects are considered.

Introduction: The hypertensive double-transgenic (dTG) rat strain, expressing human renin and angiotensinogen, develops severe hypertension and organ damage and 50% of individuals die by 7 weeks of age. Here, we characterise a variation of this model in which animals present stable hypertension.Materials and methods: The effect of renin—angiotensin system blockers on blood pressure was determined with adult dTG rats treated with enalapril from 3 to 12 weeks of age. Tissue expression levels of renin and angiotensinogen were determined in dTG rats and rhesus monkeys by quantitative PCR.Results: Upon withdrawal from enalapril, mean arterial pressure (MAP) rose to 160—180 mmHg, with 95% of the female dTG rats surviving for 6 to 12 months, In Sprague-Dawley (SD) rats and rhesus monkeys, renin mRNA was absent or weakly expressed in most tissues, except for the kidneys and adrenals. In dTG rats, human renin expression was high in many additional tissues. The expression of human angiotensinogen in dTG rats followed a similar tissue pattern to SD and rhesus monkey angiotensinogen. Oral dosing of aliskiren, enalapril or losartan provided a similar maximal reduction in MAP and duration of efficacy in telemetrised dTG rats.Conclusions: Enalapril-pretreated dTG rats are suitable for long-term MAP monitoring and sequential evaluation of human renin inhibitors.

An SAR campaign aimed at decreasing the overall lipophilicity of renin inhibitors such as 1 is described herein. It was found that replacement of the northern appendage in 1 with an N-methyl pyridone and subsequent re-optimization of the benzyl amide handle afforded compounds with in vitro and in vivo profiles suitable for further profiling. An unexpected CV toxicity in dogs observed with compound 20 led to the employment of a time and resource sparing rodent model for in vivo screening of key compounds. This culminated in the identification of compound 31 as an optimized renin inhibitor.

A human cDNA clone encoding a nonreceptor protein-tyrosine-phosphatase (PTP) has been isolated and sequenced. The 2.1 kilobase pair cDNA encodes for a 593 amino acid protein that contains a single tyrosine phosphatase catalytic domain at the C-terminus. At the N-terminus the protein has two adjacent copies of Src homology region (SH2 domain) which show 61% and 73% identity at the amino acid level to the SH2 domains of the human PTP1C and Drosophila corkscrew protein, respectively. The overall homology between SH-PTP2 and PTP1C or to corkscrew protein is 58%. When this protein (or its catalytic domain) was expressed in E. coli as a glutathione-S-transferase fusion protein tyrosine-phosphatase activity was detected in bacterial cell extracts. Site-directed mutation made at the conserved cysteine (459) residue to serine within the highly conserved VHCXAGXXR sequence in the PTP catalytic domain resulted in complete loss of enzymatic activity demonstrating the importance of this cysteine residue in catalysis. Northern blot analysis showed that SH-PTP2 is expressed as a 6.5 kilobase mRNA in a number of fetal and adult human tissues and cell lines. The highest levels of its mRNA were detected in fetal brain and in adult heart tissue. The identification of SH-PTP2 along with PTP1C and corkscrew protein suggest that there exist a family of nonreceptor PTP containing SH2-domain which will participate in specific signal transduction pathways involving tyrosine phosphorylation-dephosphorylation.

Renin is the first enzyme in the renin-angiotensin-aldosterone system which is the principal regulator of blood pressure and hydroelectrolyte balance. Previous studies suggest that cathepsin B is the activator of the prorenin zymogen. Here, we show no difference in plasma renin activity, or mean arterial blood pressure between wild-type and cathepsin B knockout mice. To account for potential gene compensation, a potent, selective, reversible cathepsin B inhibitor was developed to determine the role of cathepsin B on prorenin processing in rats. Pharmacological inhibition of cathepsin B in spontaneously hypertensive and double transgenic rats did not result in a reduction in renal mature renin protein levels or plasma renin activity. We conclude that cathepsin B does not play a significant role in this process in rodents.

The structure-activity relationship of a novel series of 8-biarylnaphthyridinones acting as type 4 phosphodiesterase (PDE4) inhibitors for the treatment of long-term memory loss and mild cognitive impairment is described herein. The manuscript describes a new paradigm for the development of PDE4 inhibitor targeting CNS indications. This effort led to the discovery of the clinical candidate MK-0952, an intrinsically potent inhibitor (IC(50)=0.6 nM) displaying limited whole blood activity (IC(50)=555 nM). Supporting in vivo results in two preclinical efficacy tests and one test assessing adverse effects are also reported. The comparative profiles of MK-0952 and two other Merck compounds are described to validate the proposed hypothesis.

Substituted 8-arylquinoline analogs bearing alkyl-linked side chain were identified as potent inhibitors of type 4 phophodiesterase. These compounds address the potential liabilities of the clinical candidate L-454560. The pharmacokinetic profile of the best analogs and the in vivo efficacy in an ovalbumin-induced bronchoconstriction assay in conscious guinea pigs are reported.

Plasma renin activity (PRA) is a well-established biomarker for assessing the efficacy of various antihypertensive agents such as direct renin inhibitors, angiotensin receptor blockers, and angiotensin-converting enzyme inhibitors (ACEIs). PRA measurements are obtained through the detection and quantification of angiotensin I (Ang I) produced by the action of renin on its natural substrate angiotensinogen. The most accepted and reproducible method for PRA measurement uses an antibody capture Ang I methodology that employs specific antibodies that recognize and protect Ang I against angiotensinase activities contained in plasma. The amount of Ang I is then quantified by either radioimmunoassay (RIA) or enzyme immunoassay (EIA). In the current report, we describe the optimization of a novel homogeneous immunoassay based on the AlphaScreen technology for the detection and quantification of antibody-captured Ang I using AlphaLISA acceptor beads in buffer and in the plasma of various species (human, rat, and mouse). Ex vivo measurements of renin activity were performed using 10 microl or less of a reaction mixture, and concentrations as low as 1 nM Ang I were quantified. Titration curves obtained for the quantification of Ang I in buffer and plasma gave similar EC(50) values of 5.6 and 14.4 nM, respectively. Both matrices generated an equivalent dynamic range that varies from approximately 1 to 50 nM. Renin inhibitors have been successfully titrated and IC(50) values obtained correlated well with those obtained using EIA methodology (r(2)=0.80). This assay is sensitive, robust, fast, and less tedious than measurements performed using nonhomogeneous EIA. The AlphaLISA methodology is homogeneous, does not require wash steps prior to the addition of reagents, and does not generate radioactive waste.

The structure-activity relationship of a novel series of 8-biarylquinolines acting as type 4 phosphodiesterase (PDE4) inhibitors is described herein. Prototypical compounds from this series are potent and non-selective inhibitors of the four distinct PDE4 (IC(50)10 nM) isozymes (A-D). In a human whole blood in vitro assay, they inhibit (IC(50)0.5 microM) the LPS-induced release of the cytokine TNF-alpha. Optimized inhibitors were evaluated in vivo for efficacy in an ovalbumin-induced bronchoconstriction model in conscious guinea pigs. Their propensity to produce an emetic response was evaluated by performing pharmacokinetic studies in squirrel monkeys. This work has led to the identification of several compounds with excellent in vitro and in vivo profiles, including a good therapeutic window of efficacy over emesis.

Potent inhibitors of the human PDE IV enzyme are described. Substituted 8-arylquinoline analogs bearing nitrogen-linked side chain were identified as potent inhibitors based on the SAR described herein. The pharmacokinetic profile of the best analog and the in vivo efficacy in an ovalbumin-induced bronchoconstriction assay in conscious guinea pigs are reported.

Ibudilast ophthalmic solution exhibited an improved clinical efficacy over cromoglycate in the treatment of allergic conjunctivitis. To further characterize its principal mode of action, the phosphodiesterase (PDE) inhibitory profile of ibudilast has been examined using human recombinant enzymes. Ibudilast, but not the other commonly used anti-allergic ophthalmic solutions including cromoglycate, ketotifen, tranilast and levocabastine, potently inhibits purified human PDE4A, 4B, 4C and 4D with IC50 values at 54, 65, 239 and 166 nM, respectively. Ibudilast effectively blocks lipopolysaccharide (LPS)-induced tumor necrosis factor (TNFalpha, IC50 = 6.2 microM) and N-formyl-Met-Leu-Phe (fMLP)-induced leukotriene (LT) B4 biosynthesis (IC50 = 2.5 microM) in human whole blood, which are 3 and 6-fold more potent than cilomilast, respectively. The attenuated inflammatory and allergic responses from the potent and preferential PDE4 inhibition of ibudilast may have contributed significantly to its beneficial pharmacological responses and distinguishes ibudilast from the other ophthalmic solutions in the treatment of ocular allergy.

The diseases of cystic fibrosis, chronic obstructive pulmonary disease (COPD), and chronic bronchitis are characterized by mucus-congested and inflamed airways. Anti-inflammatory agents that can simultaneously restore or enhance mucociliary clearance through cystic fibrosis transmembrane conductance regulator (CFTR) activation may represent new therapeutics in their treatment. Herein, we report the activation of CFTR-mediated chloride secretion by phosphodiesterase (PDE) 4 inhibitors in T84 monolayer using (125)I anion as tracer. In the absence of forskolin, the iodide secretion was insensitive to PDE4 inhibitor L-826,141 [4-[2-(3,4-bis-difluoromethoxyphenyl)-2-[4-(1,1,1,3,3,3-hexafluoro-2-hydroxypropan-2-yl)phenyl]-ethyl]-3-methylpyridine-1-oxide], roflumilast, or to PDE3 inhibitor trequinsin. However, these inhibitors potently augmented iodide secretion after forskolin stimulation, with efficacy coupled to the activation states of adenylyl cyclase. The iodide secretion from PDE3 or PDE4 inhibition was characterized at first by a prolonged efflux duration, followed by progressively elevated peak efflux rates at higher inhibitor concentrations. Paralleled with an increased phosphor-cAMP response element-binding protein formation, the CFTR activation dissociated from a global cAMP elevation and was blocked by H89 [N-[2-((p-bromocinnamyl)amino)ethyl]-5-isoquinolinesulfonamide]. 2-(4-Fluorophenoxy)-N-[(1S)-1-(4-methoxyphenyl)ethyl]nicotinamide, a stereoselective PDE4D inhibitor, augmented iodide efflux more efficiently than its less potent (R)-isomer. The peak efflux from maximal PDE4 and PDE3 inhibition matched that from full adenylyl cyclase activation. These data suggest that PDE3 and PDE4 (mainly PDE4D) form the major cAMP diffusion barrier in T84 cells to ensure a compartmentalized CFTR signaling. Together with their potent anti-inflammatory properties, the potentially enhanced airway mucociliary clearance from CFTR activation may have contributed to the efficacy of PDE4 inhibitors in COPD and asthmatic patients. PDE4 inhibitors may represent new opportunities to combat cystic fibrosis and other respiratory diseases in future.

A combination of pharmacological and genetic approaches was used to determine the role of type 4 cAMP-specific cyclic nucleotide phosphodiesterase 4 (PDE4) in reversing α2-adrenoceptor–mediated anesthesia, a behavioral correlate of emesis in non-vomiting species. Among the family-specific PDE inhibitors, PDE4 inhibitors reduced the duration of xylazine/ketamine–induced anesthesia in mice, with no effect on pentobarbital-induced anesthesia. The rank order of the PDE4 inhibitors tested was 6-(4-pyridylmethyl)-8-(3-nitrophenyl)quinoline (PMNPQ) > (R)-rolipram > (S)-rolipram >> (R)-N-{4-[1-(3-cyclopentyloxy-4-methoxyphenyl)-2-(4-pyridyl)ethyl]phenyl}N′-ethylurea (CT-2450). The specific roles of PDE4B and PDE4D in this model were studied using mice deficient in either subtype. PDE4D-deficient mice, but not PDE4B-deficient mice, had a shorter sleeping time than their wild-type littermates under xylazine/ketamine–induced anesthesia, but not under that induced with pentobarbital. Concomitantly, rolipram-sensitive PDE activity in the brain stem was decreased only in PDE4D-deficient mice compared with their wild-type littermates. While PMNPQ significantly reduced the xylazine/ketamine–induced anesthesia period in wild-type mice and in PDE4B-null mice, it had no effect in PDE4D-deficient mice. These findings strongly support the hypothesis that inhibition of PDE4D is pivotal to the anesthesia-reversing effect of PMNPQ and is likely responsible for emesis induced by PDE4 inhibitors.

Type 4 phosphodiesterases (PDE4s) are metallohydrolases that catalyze the hydrolysis of cAMP to AMP. At the bottom of its active site lie two divalent metal ions in a binuclear motif which are involved in both cAMP binding and catalysis [(2000) Science 288, 1822-1825; (2000) Biochemistry 39, 6449-6458]. Using a SPA-based equilibrium [(3)H]rolipram binding assay, we have determined that Mg(2+), Mn(2+), and Co(2+) all mediated a high-affinity (K(d) between 3 and 8 nM) and near stoichiometric (R)-rolipram binding to PDE4. In their absence, (R)-rolipram binds stoichiometrically to the metal ion-free apoenzyme with a K(d) of approximately 150 nM. The divalent cation dose responses in mediating the high-affinity rolipram/PDE4 interaction mirror their efficacy in catalysis, suggesting that both metal ions of the holoenzyme are involved in mediating the high-affinity (R)-rolipram/PDE4 interaction. The specific rolipram binding to the apo- and holoenzyme is differentially displaced by cAMP, AMP, and other inhibitors, providing a robust tool to dissect the components of metal ion-dependent and independent PDE4/ligand interactions. cAMP binds to the holoenzyme with a K(s) of 1.9 microM and nonproductively to the apoenzyme with a K(d) of 179 microM. In comparison, AMP binds to the holo- and apoenzyme with K(d) values of 7 and 11 mM, respectively. The diminished Mg(2+)-dependent component of AMP binding to PDE4 suggests that most of the Mg(2+)/phosphate interaction in the cAMP/PDE4 complex is disrupted upon the hydrolysis of the cyclic phosphoester bond, leading to the rapid release of AMP.

The type 4 cAMP-specific phosphodiesterases (PDE4s) are Mg(2+)-dependent hydrolases that catalyze the hydrolysis of 3', 5'-cAMP to AMP. Previous studies indicate that PDE4 exists in two conformations that bind the inhibitor rolipram with affinities differing by more than 100-fold. Here we report that these two conformations are the consequence of PDE4 binding to its metal cofactor such as Mg(2+). Using a fluorescence resonance energy transfer (FRET)-based equilibrium binding assay, we identified that L-791,760, a fluorescent inhibitor, binds to the apoenzyme (free enzyme) and the holoenzyme (enzyme bound to Mg(2+)) with comparable affinities (K(d) approximately 30 nM). By measuring the displacement of the bound L-791,760, we have also identified that other inhibitors bind differentially with the apoenzyme and the holoenzyme depending upon their structure. CDP-840, SB-207499, and RP-73401 bind preferentially to the holoenzyme. The conformational-sensitive inhibitor (R)-rolipram binds to the holoenzyme and apoenzyme with affinities (K(d)) of 5 and 300 nM, respectively. In contrast to its high affinity (K(d) approximately 2 microM) and active holoenzyme complex, cAMP binds to the apoenzyme nonproductively with a reduced affinity (K(d) approximately 170 microM). These results demonstrate that cofactor binding to PDE4 is responsible for eliciting its high-affinity interaction with cAMP and the activation of catalysis.

Arachidonyl trifluoromethyl ketone (AACOCF3) is a potent and selective slow binding inhibitor of the 85-kDa cytosolic phospholipase A2 (cPLA2) (Street, I. P., Lin, H.-K., Laliberte, F., Ghomashchi, F., Wang, Z., Perrier, H., Tremblay, N. M., Huang, Z., Weech, P. K., and Gelb, M. H. (1993) Biochemistry 32, 5935-5940). AACOCF3 and a number of its structural analogues have been used to investigate the role of cPLA2 in the cellular generation of free arachidonic acid (AA) and in eicosanoid biosynthesis. AACOCF3 inhibited the release of AA from calcium ionophore-challenged U937 cells (IC50 = 8 microM, 2 x 10(6) cells ml-1) and from platelets (IC50 = 2 microM, 4 x 10(7) cells ml-1). Arachidonyl methyl ketone (AACOCH3) and AACH(OH)CF3, both of which are noninhibitory to the purified cPLA2, did not inhibit the production of AA in the ionophore-challenged cells. In addition to the release of AA, AACOCF3 also inhibited the production of 12-hydroxyeicosatetraenoic acid (12-HETE) and thromboxane B2, two of the major metabolites of AA produced by platelets. The inhibition of 12-HETE biosynthesis showed a dose dependence similar to that of AA release in ionophore-challenged platelets; however, when platelet 12-HETE production was stimulated with 10 microM AA to circumvent the PLA2-dependent step, AACOCF3 no longer inhibited the production of 12-HETE. In contrast, AACOCF3 blocked thromboxane B2 formation by both calcium ionophore- and AA-challenged platelets, indicating that the compound affects the cyclooxygenase pathway in addition to AA release. The crude cytosol and membrane fractions from platelets were assayed for calcium-dependent and calcium-independent PLA2 activities and for the susceptibility of each to inhibition by AACOCF3. At AACOCF3 concentrations as high as 10 mol %, only one of the observed PLA2 activities was inhibited by more than 25%. The AACOCF3-susceptible PLA2 (77% inhibition at 1.6 mol %) was found in the cytosolic platelet fraction and showed the functional characteristics of the cPLA2. These results suggest that the cPLA2 plays an important role in the generation of free AA for 12-HETE biosynthesis in platelets.

The PDE4 catalytic machinery comprises, in part, two divalent cations in a binuclear motif. Here we report that PDE4A4 expressed in Sf9 cells exhibits a biphasic Mg2+ dose–response (EC50 of ∼0.15 and >10 mM) in catalyzing cAMP hydrolysis. In vitro phosphorylation of PDE4A4 by the PKA-catalytic subunit increases the enzyme’s sensitivity to Mg2+, leading to 4-fold increased cAMP hydrolysis without affecting its Km. The phosphorylation also increases the potencies of (R)- and (S)-rolipram without affecting CDP-840 and SB-207499. The results support that modulating the cofactor binding affinity of PDE4 represents a mechanism for regulating its activity.