A Cautionary Tale: Quantitative LC-HRMS Analytical Procedures for the Analysis of N-Nitrosodimethylamine in Metformin.

A private testing laboratory reported in a Citizen Petition (CP) to FDA that 16 of 38 metformin drug products they tested had N-nitrosodimethyl amine (NDMA) amounts above the allowable intake (AI) of 96 ng/day. Because the FDA had been monitoring drugs for nitrosamines, orthogonal analytical procedures had been developed, validated and applied to detect the following nitrosamines in metformin drug products (if present): (i) NDMA (with a dedicated method) or (ii) NDMA (with a second confirmatory method), N-nitroso-diethylamine (NDEA), N-ethyl-N-nitroso-2-propanamine (NEIPA), N-nitroso-diisopropylamine (NDIPA), N-nitroso-di-n-propylamine (NDPA), N-nitroso-methylphenylamine (NMPA), N-nitroso-di-n-butylamine (NDBA) and N-nitroso-N-methyl-4-aminobutyric acid (NMBA). In contrast to the private laboratory results, FDA testing on the same set of 38 samples with orthogonal procedures observed amounts over the AI in only 8 of the 38 products and generally observed lower values than reported by the private testing laboratory. As described here, the investigation into the cause of the discrepancy revealed that N,N-dimethylformamide (DMF) can interfere with NDMA measurements. The data showed that the use of sufficient mass accuracy in the data acquisition and appropriate mass tolerance setting in the data processing to assure the selectivity of mass spectrometry measurements of NDMA in the presence of co-eluting DMF was necessary to prevent overestimation of the level of NDMA in metformin drug products. Overall, care should be taken to assure the necessary specificity in analytical procedures for adequate assessment of the nitrosamine level in drug products that also contain DMF or other potential interfering substances.

PF4-HIT antibody (KKO) complexes activate broad innate immune and inflammatory responses.

INTRODUCTION: Heparin-induced thrombocytopenia (HIT) is an immune-mediated complication of heparin anticoagulation therapy resulting in thrombocytopenia frequently accompanied by thrombosis. Current evidence suggests that HIT is associated with antibodies developed in response to multi-molecular complexes formed by platelet factor 4 (PF4) bound to heparin or cell surface glycosaminoglycans. These antibody complexes activate platelets and monocytes typically through FcγRIIA receptors increasing the production of PF4, inflammatory mediators, tissue factor and thrombin. The influence of underlying events in HIT including complex-induced pro-inflammatory cell activation and structural determinants leading to local inflammatory responses are not fully understood. METHODS: The stoichiometry and complex component requirements were determined by incubating fresh peripheral blood mononuclear cells (PBMC) with different concentrations of unfractionated heparin (H), low molecular weight heparin (LMWH), PF4- and anti-PF4-H complex antibodies (KKO). Cytokine mRNA or protein were measured by qRT-PCR or Meso Scale Discovery technology, respectively. Gene expression profile analysis for 594 genes was performed using Nanostring technology and analyzed using Ingenuity Pathway Analysis software. RESULTS AND CONCLUSIONS: The data show that antibodies magnify immune responses induced in PBMCs by PF4 alone or in complex with heparin or LMWH. We propose that following induction of HIT antibodies by heparin-PF4 complexes, binding of the antibodies to PF4 is sufficient to induce a local pro-inflammatory response which may play a role in the progression of HIT. In vitro assays using PBMCs may be useful in characterizing local inflammatory and innate immune responses induced by HIT antibodies in the presence of PF4 and different sources of heparins. FDA DISCLAIMER: The findings and conclusions in this article are solely the responsibility of the authors and are not being formally disseminated by the Food and Drug Administration. Thus, they should not be construed to represent any Agency determination or policy.

Heparin and homogeneous model heparin oligosaccharides form distinct complexes with protamine: Light scattering and zeta potential analysis.

Large multimolecular complexes of heparin with positively charged proteins such as platelet factor 4 (PF4) or protamine can initiate immune responses associated with heparin use in patients, including the most significant adverse event, heparin-induced thrombocytopenia (HIT). Current evidence suggests that platelet-activating antibodies that recognize large multi-molecular complexes (300-700kDa) of PF4 bound to heparin cause HIT [1] and in very rare cases anti-protamine-heparin antibodies can induce thrombocytopenia [2]. Heparin is administered as a mixture of sulfated glycosaminoglycans of variable lengths and sulfation levels. To date the potential impact of chain length, sulfation level and impurities on the formation, size and immunogenicity of heparin-protamine complexes has not been addressed due to the lack of purified, homogenous heparin chains for testing purposes. Here, a set of well-characterized model heparin oligosaccharides was used with protamine sulfate to evaluate the physicochemical properties of the resulting complexes. Hydrodynamic radii and zeta potential profiles of heparin-protamine complexes were observed to be dependent upon the sulfation location, size and concentration of the model heparin oligosaccharides. The well-characterized oligosaccharide-protamine complexes analyzed in this work will be useful for establishing links between heparin-protamine complex physiochemical attributes to their potential to illicit cellular immunogenicity.

Analytical techniques and bioactivity assays to compare the structure and function of filgrastim (granulocyte-colony stimulating factor) therapeutics from different manufacturers.

The FDA has approved more than 100 protein and peptide drugs with hundreds more in the pipeline (Lanthier et al. in Nat Rev Drug Discov 7(9):733-737, 2008). Many of these originator biologic products are now coming off patent and are being manufactured by alternate methods than the innovator as follow-on drugs. Because changes to the production method often lead to subtle differences (e.g., degradation products, different posttranslational modifications or impurities) in the therapeutic (Schiestl et al. in Nat Biotechnol 29(4):310-312, 2011), there is a critical need to define techniques to test and insure the quality of these drugs. In addition, the emergence of protein therapeutics manufactured by unapproved methodologies presents an ongoing and growing regulatory challenge. In this work, high-resolution mass spectrometry was used to determine the presence or absence of posttranslational modifications for one FDA-approved and three foreign-sourced, unapproved filgrastim products. Circular dichroism (CD) was used to compare the secondary structure and probe the temperature stability of these products. Native 2D (1)H,(15)N-heteronuclear singular quantum coherence (HSQC) NMR test was applied to these samples to compare the higher-order structure of the four products. Finally, a cell proliferation assay was performed on the filgrastims to compare their bioactivity, and stressed filgrastim was tested in the bioassay to better understand the effects of changes in protein structure on activity. The results showed that orthogonal approaches are capable of characterizing the physiochemical properties of this protein drug and assessing the impact of structural changes on filgrastim purity and potency.

Characterization of currently marketed heparin products: key tests for LMWH quality assurance.

During the 2007-2008 heparin crisis it was found that the United States Pharmacopeia (USP) testing monograph for heparin sodium or low molecular weight heparins did not detect the presence of the contaminant, oversulfated chondroitin sulfate (OSCS). In response to this concern, new tests and specifications were developed by the Food and Drug Administration (FDA) and USP and put in place to detect not only the contaminant OSCS, but also to improve assurance of quality and purity of these drug products. The USP monographs for the low molecular weight heparins (LMWHs) approved for use in the United States (dalteparin, tinzaparin and enoxaparin) are also undergoing revision to include many of the same tests used for heparin sodium, including; one-dimensional (1D) 500 MHz (1)H NMR, SAX-HPLC, percent galactosamine in total hexosamine and anticoagulation time assays with purified Factor IIa or Factor Xa. These tests represent orthogonal approaches for heparin identification, measurement of bioactivity and for detection of process impurities or contaminants in these drug products. Here we describe results from a survey of multiple lots from three types of LMWHs in the US market which were collected after the 2009 heparin sodium monograph revision. In addition, innovator and generic versions of formulated enoxaparin products purchased in 2011 are compared using these tests and found to be highly similar within the discriminating power of the assays applied.

Analyses of marketplace tacrolimus drug product quality: bioactivity, NMR and LC-MS.

Tacrolimus (FK506) is a potent, narrow therapeutic index, immunosuppressive drug used to avoid organ rejection in patients that have undergone organ transplantation. Recent clinical reports suggested a significant reduction in the tacrolimus concentration/dose ratio in the plasma of liver and kidney recipients when the reference listed drug was substituted with a generic drug. In response to these concerns about switching between tacrolimus from different approved manufacturers during treatment, the FDA initiated purity, potency and quality studies of the innovator and generic tacrolimus products available in the US marketplace. A combination of analytical methods, including mass spectrometry (LC-MS), nuclear magnetic resonance (NMR) and bioactivity assay were developed and validated to assess the quality of tacrolimus. These tests measured the identity, impurities and activity of tacrolimus from active pharmaceutical ingredient (API) sources and with formulated drug product from five different approved manufactures. In addition, some testing was performed on tacrolimus capsules obtained from a non US approved Indian source. The data obtained showed no discernible difference in the impurity profiles and potency between the generic and innovator tacrolimus products.

Characterization of currently marketed heparin products: adverse event relevant bioassays.

The polyanion oversulfated chondroitin sulfate (OSCS) was identified as a contaminant in heparin products and was associated with severe hypotensive responses and other symptoms in patients receiving the drug. The OSCS associated adverse reactions were attributed to activation of the contact system via the plasma mediator, activated factor XII (FXIIa), which triggers kallikrein (KK) activity. Unlike heparin alone, OSCS, is able to activate FXII in plasma and stably bind to FXIIa enhancing plasma KK activity and the induction of vasoactive mediators such as bradykinin (BK), C3a and C5a. Similarly OSCS can interfere with heparin neutralization by the polycationic drug protamine. Here, we assess heparin (heparin sodium, dalteparin, tinzaparin or enoxaparin)-protamine complex formation and plasma based bioassays of KK, BK and C5a in a 96-well plate format. We establish the normal range of variation in the optimized bioassays across multiple lots from 9 manufacturers. In addition, because other oversulfated (OS) glycosaminoglycans (GAGs) besides OSCS could also serve as possible economically motivated adulterants (EMAs) to heparin, we characterize OS-dermatan sulfate (OSDS), OS-heparan sulfate (OSHS) and their native forms in the same assays. For the protamine test, OS-GAGs could be distinguished from heparin. For the KK assay, OSCS and OSDS were most potent followed by OSHS, and all had similar efficacies. Finally, OSDS had a greater efficacy in the C5a and BK assays followed by OSCS then OSHS. These data established the normal range of response of heparin products in these assays and the alteration in the responses in the presence of possible EMAs.

Differential kinetics and inhibition of purified recombinant tyrosine kinase 2 (TYK-2) and its catalytic domain JH-1.

The Janus kinase (JAK) family consists of four members: JAK-1, -2, -3 and tyrosine kinase 2 (TYK-2). Recent work suggests that cytokine signaling through TYK-2 may play a critical role in a number of inflammatory processes. We recently described the purification and characterization of phosphorylated isoforms of the TYK-2 kinase domain (TYK-2 KD) and its high resolution 3D structure in the presence of inhibitors. We now report the expression and a two-step purification procedure for the doubly tagged full-length construct, H6-FL-TYK-2-FLAG, and examine its properties compared to those of TYK-2 KD. In the presence of ATP and a peptide substrate, H6-FL-TYK-2-FLAG showed a marked lag in phosphopeptide product formation, while TYK-2 KD showed no such lag. This lag could be eliminated by ATP pretreatment, suggesting that the H6-FL-TYK-2-FLAG enzyme was activated by phosphorylation. The potencies of several nanomolar inhibitors were similar for TYK-2 KD and H6-FL-TYK-2-FLAG. However, these same inhibitors were about 1000 times less potent inhibiting the autophosphorylation of H6-FL-TYK-2-FLAG than they were inhibiting the phosphorylation of a peptide substrate modeled after the activation loop sequence of TYK-2. This intriguing result suggests that autophosphorylation and, thus, activation of H6-FL-TYK-2-FLAG is relatively insensitive to inhibition and that present inhibitors act to inhibit TYK-2 subsequent to activation. Inhibition of TYK-2 autophosphorylation may represent a new area of investigation for the JAK family.

Quality assessment of U.S. marketplace vancomycin for injection products using high-resolution liquid chromatography-mass spectrometry and potency assays.

In response to a published concern about the potency and quality of generic vancomycin products, the United States Food and Drug Administration investigated a small sampling of the vancomycin products available in North America with regard to purity, content, and potency. To facilitate identification of impurities, a new liquid chromatography method was developed using high-resolution mass spectrometry in addition to diode array detection to characterize impurities in several commercial products. Furthermore, a microbiological assay was utilized to link the analytical profiles with an in vitro potency. All products tested met the quality specifications outlined in the United States Pharmacopeia (USP) (vancomycin hydrochloride for injection monograph) for impurities and potency (USP, Vancomycin hydrochloride for injection. United States Pharmacopeia and National Formulary, vol USP 34-NF 29, 2011).

Characterization of currently marketed heparin products: analysis of molecular weight and heparinase-I digest patterns.

We evaluated polyacrylamide gel electrophoresis (PAGE) and size exclusion chromatography coupled with multi-angle laser light scattering (SEC-MALLS) approaches to determine weight-average molecular weight (M(w)) and polydispersity (PD) of heparins. A set of unfractionated heparin sodium (UFH) and low-molecular-weight heparin (LMWH) samples obtained from nine manufacturers which supply the US market were assessed. For SEC-MALLS, we measured values for water content, refractive index increment (dn/dc), and the second virial coefficient (A(2)) for each sample prior to molecular weight assessment. For UFH, a mean ± standard deviation value for M(w) of 16,773 ± 797 was observed with a range of 15,620 to 18,363 (n = 20, run in triplicate). For LMWHs by SEC-MALLS, we measured mean M(w) values for dalteparin, tinzaparin, and enoxaparin of 6,717 ± 71 (n = 4), 6,670 ± 417 (n = 3), and 3,959 ± 145 (n = 3), respectively. PAGE analysis of the same UFH, dalteparin, tinzaparin, and enoxaparin samples showed values of 16,135 ± 643 (n = 20), 5,845 ± 45 (n = 4), 6,049 ± 95 (n = 3), and 4,772 ± 69 (n = 3), respectively. These orthogonal measurements are the first M(w) results obtained with a large heparin sample set on product being marketed after the heparin crisis of 2008 changed the level of scrutiny of this drug class. In this study, we compare our new data set to samples analyzed over 10 years earlier. In addition, we found that the PAGE analysis of heparinase digested UFH and neat LMWH samples yield characteristic patterns that provide a facile approach for identification and assessment of drug quality and uniformity.

Detection of possible economically motivated adulterants in heparin sodium and low molecular weight heparins with a colorimetric microplate based assay.

Recently, we described a 96-well plate format assay for visual detection of oversulfated chondroitin sulfate A (OSCS) contamination in heparin samples based on a water-soluble cationic polythiophene polymer (3-(2-(N-(N'-methylimidazole))ethoxy)-4-methylthiophene (LPTP)) and heparinase digestion of heparin. Here, we establish the specificity of the LPTP/heparinase test with a unique set of reagents that define the structural requirements for significant LPTP chemosensor color change. For example, we observed a biphasic behavior of larger shifts to the red in the UV absorbance spectra with decreasing average molecular weight of heparin chains with a break below 12-mer chain lengths. In addition, the oversulfation of chondroitin sulfate A (CSA) to a partially (PSCS) or fully (OSCS) sulfated form caused progressively less red shift of LPTP solutions. Furthermore, glycosaminoglycans (GAGs) containing glucuronic acid caused distinct spectral patterns compared to iduronic acid containing GAGs. We applied the LPTP/heparinase test to detection of OSCS (≥0.03% (w/w) visually or 0.01% using a plate reader) in 10 µg amounts of low molecular weight heparins (LMWHs; i.e. dalteparin, tinzaparin, or enoxaparin). Furthermore, because other oversulfated GAGs are possible economically motivated adulterants (EMAs) in heparin sodium, we tested the capacity of the LPTP/heparinase assay to detect oversulfated dermatan sulfate (OSDS), heparin (OSH), and heparan sulfate (OSHS). These potential EMAs were visually detectable at a level of ∼0.1% when spiked into heparin sodium. We conclude that the LPTP/heparinase test visually detects oversulfated GAGs in heparin sodium and LMWHs in a format potentially amenable to high-throughput screening.

Sensitive detection of oversulfated chondroitin sulfate in heparin sodium or crude heparin with a colorimetric microplate based assay.

In this work we describe a 96-well microplate assay for oversulfated chondroitin sulfate A (OSCS) in heparin, based on a water-soluble cationic polythiophene polymer (3-(2-(N-(N'-methylimidazole))ethoxy)-4-methylthiophene (LPTP)) and heparinase digestion of heparin. The assay takes advantage of several unique properties of heparin, OSCS, and LPTP, including OSCS inhibition of heparinase I and II activity, the molecular weight dependence of heparin-LPTP spectral shifts, and the distinct association of heparin fragments and OSCS to LPTP. These factors combine to enable detection of the presence of 0.003% w/w spiked OSCS in 10 µg of heparin sodium active pharmaceutical ingredient (API) using a plate reader and with visual detection to 0.1% levels. The same detection limit for OSCS was observed in the presence of 10% levels of dermatan sulfate (DS) or chondroitin sulfate A (CSA) impurities. In addition, we surveyed a selection of crude heparin samples received by the agency in 2008 and 2009 to determine average and extreme DS, CSA, and galactosamine weight percent levels. In the presence of these impurities and the variable heparin content in the crude heparin samples, spiked OSCS was reliably detected to the 0.1% w/w level using a plate reader. Finally, authentically OSCS contaminated heparin sodium API and crude samples were distinguished visually by color from control samples using the LPTP/heparinase test.

Aminopyridinecarboxamide-based inhaled IKK-2 inhibitors for asthma and COPD: Structure-activity relationship.

Installation of sites for metabolism in the lead compound PHA-767408 was the key focus of the IKK-2 inhaled program. This paper reports our efforts to identify a novel series of aminopyridinecarboxamide-based IKK-2 inhibitors, which display low nanomolar potency against IKK-2 with long duration of action (DOA), and metabolically labile to phase I and/or phase II metabolizing enzymes with potential capability for multiple routes of clearance. Several compounds have demonstrated their potential usefulness in the treatment of asthma and chronic obstructive pulmonary disease (COPD).

Structural and thermodynamic characterization of the TYK2 and JAK3 kinase domains in complex with CP-690550 and CMP-6.

Janus kinases (JAKs) are critical regulators of cytokine pathways and attractive targets of therapeutic value in both inflammatory and myeloproliferative diseases. Although the crystal structures of active JAK1 and JAK2 kinase domains have been reported recently with the clinical compound CP-690550, the structures of both TYK2 and JAK3 with CP-690550 have remained outstanding. Here, we report the crystal structures of TYK2, a first in class structure, and JAK3 in complex with PAN-JAK inhibitors CP-690550 ((3R,4R)-3-[4-methyl-3-[N-methyl-N-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)amino]piperidin-1-yl]-3-oxopropionitrile) and CMP-6 (tetracyclic pyridone 2-t-butyl-9-fluoro-3,6-dihydro-7H-benz[h]-imidaz[4,5-f]isoquinoline-7-one), both of which bind in the ATP-binding cavities of both JAK isozymes in orientations similar to that observed in crystal structures of JAK1 and JAK2. Additionally, a complete thermodynamic characterization of JAK/CP-690550 complex formation was completed by isothermal titration calorimetry, indicating the critical role of the nitrile group from the CP-690550 compound. Finally, computational analysis using WaterMap further highlights the critical positioning of the CP-690550 nitrile group in the displacement of an unfavorable water molecule beneath the glycine-rich loop. Taken together, the data emphasize the outstanding properties of the kinome-selective JAK inhibitor CP-690550, as well as the challenges in obtaining JAK isozyme-selective inhibitors due to the overall structural and sequence similarities between the TYK2, JAK1, JAK2 and JAK3 isozymes. Nevertheless, subtle amino acid variations of residues lining the ligand-binding cavity of the JAK enzymes, as well as the global positioning of the glycine-rich loop, might provide the initial clues to obtaining JAK-isozyme selective inhibitors.

Expression, purification, and characterization of TYK-2 kinase domain, a member of the Janus kinase family.

The Janus kinase family consists of four members: JAK-1, -2, -3 and TYK-2. While JAK-2 and JAK-3 have been well characterized biochemically, there is little data on TYK-2. Recent work suggests that TYK-2 may play a critical role in the development of a number of inflammatory processes. We have carried out a series of biochemical studies to better understand TYK-2 enzymology and its inhibition profile, in particular how the TYK-2 phosphorylated forms differ from each other and from the other JAK family members. We have expressed and purified milligram quantities of the TYK-2 kinase domain (KD) to high purity and developed a method to separate the non-, mono- (pY(1054)) and di-phosphorylated forms of the enzyme. Kinetic studies (k(cat(app))/K(m(app))) indicated that phosphorylation of the TYK-2-KD (pY(1054)) increased the catalytic efficiency 4.4-fold compared to its non-phosphorylated form, while further phosphorylation to generate the di-phosphorylated enzyme imparted no further increase in activity. These results are in contrast to those obtained with the JAK-2-KD and JAK-3-KD, where little or no increase in activity occurred upon mono-phosphorylation, while di-phosphorylation resulted in a 5.1-fold increase in activity for the JAK-2-KD. Moreover, ATP-competitive inhibitors demonstrated 10-30-fold shifts in potency (K(i(app))) as a result of the TYK-2-KD phosphorylation state, while the shifts for JAK-3-KD were only 2-3-fold and showed little or no change for JAK-2-KD. Thus, the phosphorlyation state imparted differential effects on both activity and inhibition within the JAK family of kinases.

Aminopyridinecarboxamide-based inhibitors: Structure-activity relationship.

Series of aminopyridinecarboxamide-based inhibitors were synthesized and tested against human recombinant IKK-2 and in IL-1beta stimulated synovial fibroblasts. The 2-amino-5-chloropyridine-4-carboxamides were identified as the most potent inhibitors with improved cellular activity.

Novel tight-binding inhibitory factor-kappaB kinase (IKK-2) inhibitors demonstrate target-specific anti-inflammatory activities in cellular assays and following oral and local delivery in an in vivo model of airway inflammation.

Nuclear factor-kappaB (NF-kappaB) is one of the major families of transcription factors activated during the inflammatory response in asthma and chronic obstructive pulmonary disease. Inhibitory factor-kappaB kinase 2 (IKK-2) has been shown to play a pivotal role in cytokine-induced NF-kappaB activation in airway epithelium and in disease-relevant cells. Nevertheless, the potential toxicity of specific IKK-2 inhibitors may be unacceptable for oral delivery in chronic obstructive pulmonary disease. Therefore, local delivery to the lungs is an attractive alternative that warrants further exploration. Here, we describe potent and selective small-molecule IKK-2 inhibitors [8-(5-chloro-2-(4-methylpiperazin-1-yl)isonicotinamido)-1-(4-fluorophenyl)-4,5-dihydro-1H-benzo[g]indazole-3-carboxamide (PHA-408) and 8-(2-(3,4-bis(hydroxymethyl)-3,4-dimethylpyrrolidin-1-yl)-5-chloroisonicotinamido)-1-(4-fluorophenyl)-4,5-dihydro-1H-benzo-[g]indazole-3-carboxamide (PF-184)] that are competitive for ATP have slow off-rates from IKK-2 and display broad in vitro anti-inflammatory activities resulting from NF-kappaB pathway inhibition. Notably, PF-184 has been designed to have high systemic clearance, which limits systemic exposure and maximizes the effects locally in the airways. We used an inhaled lipopolysaccharide-induced rat model of neutrophilia to address whether inhibiting NF-kappaB activation locally within the airways would show anti-inflammatory effects in the absence of systemic exposure. PHA-408, a low-clearance compound previously shown to be efficacious orally in a rodent model of arthritis, dose-dependently attenuated inhaled lipopolysaccharide-induced cell infiltration and cytokine production. Interestingly, PF-184 produced comparable dose-dependent anti-inflammatory activity by intratracheal administration and was as efficacious as intratracheally administered fluticasone propionate (fluticasone). Together, these results support the potential therapeutic utility of IKK-2 inhibition in inflammatory pulmonary diseases and demonstrate anti-inflammatory efficacy of an inhaled IKK-2 inhibitor in a rat airway model of neutrophilia.

Pharmacokinetic and pharmacodynamic evaluation of the suitability of using fluticasone and an acute rat lung inflammation model to differentiate lung versus systemic efficacy.

Inhaled corticosteroids (ICSs) are often prescribed as the first line therapy for pulmonary diseases such as asthma. The biggest concern of using steroid therapy is the systemic side effects at high dose. To reduce the side effects, the pharmaceutical industry has been putting effort to generate new drugs with maximized topical efficacy. One of the key challenges is to differentiate efficacy from local versus systemic contribution in preclinical animal models. Fluticasone with various formulations was used as a model compound to explore the possibilities to demonstrate lung targeted efficacy by intratracheally instillation in the lipopolysaccharide induced inflammation rat model. Fluticasone formulations contained various surfactant concentrations and particle sizes to achieve lung retention and lower systemic exposure. Neutrophil infiltration in broncoalveolar lavage fluid and cytokine production in whole blood were measured to assess pulmonary efficacy versus systemic efficacy. PK/PD characterization of fluticasone with various formulations in the rat inflammation model provided an integrated approach in preclinical to evaluate lung targeted efficacy for ICS. Our study concluded that the combination of the rat LPS model and fluticasone is not suitable to use for establishing potency and dose requirement for new drug candidate designed for topical only efficacy.

A novel, highly selective, tight binding IkappaB kinase-2 (IKK-2) inhibitor: a tool to correlate IKK-2 activity to the fate and functions of the components of the nuclear factor-kappaB pathway in arthritis-relevant cells and animal models.

Nuclear factor (NF)-kappaB activation has been clearly linked to the pathogenesis of multiple inflammatory diseases including arthritis. The central role that IkappaB kinase-2 (IKK-2) plays in regulating NF-kappaB signaling in response to inflammatory stimuli has made this enzyme an attractive target for therapeutic intervention. Although diverse chemical classes of IKK-2 inhibitors have been identified, the binding kinetics of these inhibitors has limited the scope of their applications. In addition, safety assessments of IKK-2 inhibitors based on a comprehensive understanding of the pharmacokinetic/pharmacodynamic relationships have yet to be reported. Here, we describe a novel, potent, and highly selective IKK-2 inhibitor, PHA-408 [8-(5-chloro-2-(4-methylpiperazin-1-yl)isonicotinamido)-1-(4-fluorophenyl)-4,5-dihydro-1H-benzo[g]indazole-3-carboxamide]. PHA-408 is an ATP-competitive inhibitor, which binds IKK-2 tightly with a relatively slow off rate. In arthritis-relevant cells and animal models, PHA-408 suppresses inflammation-induced cellular events, including IkappaBalpha phosphorylation and degradation, p65 phosphorylation and DNA binding activity, the expression of inflammatory mediators, and joint pathology. PHA-408 was efficacious in a chronic model of arthritis with no adverse effects at maximally efficacious doses. Stemming from its ability to bind tightly to IKK-2, as a novelty, we demonstrated that PHA-408-mediated inhibition of IKK-2 activity correlated very well with its ability to modulate the fate of IKK-2 substrates and downstream transcriptional events. We ultimately directly linked IKK-2 activity ex vivo and in vivo to markers of inflammation with the inhibitor plasma concentrations. Thus, PHA-408 represents a powerful tool to further gain insight into the mechanisms by which IKK-2 regulates NF-kappaB signaling and validates IKK-2 as a therapeutic target.