Characterization of recombinant Factor IX fusion proteins enabling subcutaneous administration.

BACKGROUND: The X-linked bleeding disorder Hemophilia B, caused by mutation(s) in the coagulation factor IX (FIX) gene, leads to partial or total loss of its function requiring lifelong FIX replacement therapy. Although new recombinant FIX (rIX) therapeutics like albumin-fusion proteins (rIX-FP) enable longer plasma half-life and thus less frequent administration, the complexity of intravenous (IV) injection remains. OBJECTIVES: The study aims to characterize rIX-FP variants with anticipated enhanced specific activity, which would leverage rIX-FP's superior pharmacokinetic (PK) profile with beneficial characteristics for subcutaneous (SC) administration. METHODS: Two rIX-FP variants, R338L ("Padua-variant") and R338L/E410K, were characterized in vitro. PK profiles of FIX antigen and activity levels were evaluated in FIX-deficient mice after IV and SC administration of these variants (dosing based on antigen levels). The efficacy of the most promising variant was tested after IV and SC administration (dosing based on activity) in a tail-clip bleeding model. A marketed wildtype (WT) rIX-FP product served as the comparator. RESULTS: Both rIX-FP variants showed a 4- to 5-fold increase in specific activity in vitro compared to rIX(WT)-FP, whilst FXIa-mediated activation was the fastest for rIX(WT)-FP and rIX(R338L)-FP. Compared to rIX(WT)-FP and rIX(R338L/E410K)-FP, rIX(R338L)-FP exhibited higher FIX activity exposure after IV and SC administration, and demonstrated comparable efficacy towards rIX(WT)-FP in reducing bleeding time and blood loss in FIX-deficient mice requiring ∼4 times lower protein amount. CONCLUSIONS: rIX(R338L)-FP was shown to be a promising candidate for SC administration, exhibiting increased specific activity combined with higher activity-based exposure, and indicating efficacy at lower protein dose.

Modulation of Extravascular Binding of Recombinant Factor IX Impacts the Duration of Efficacy in Mouse Models.

BACKGROUND: There is an emerging concept that in addition to circulating coagulation factor IX (FIX), extravascular FIX contributes to hemostasis. OBJECTIVE: Our objective was to evaluate the efficacy of extravascular FIX using animal models of tail clip bleeding and ferric chloride-induced thrombosis. METHODS: Mutant rFIX proteins with described enhanced (rFIXK5R) or reduced (rFIXK5A) binding to extracellular matrix were generated and characterized using in vitro aPTT, one-stage clotting, and modified FX assays. Using hemophilia B mice, pharmacokinetic (PK) parameters and in vivo efficacy of these proteins were compared against rFIX wild-type protein (rFIXWT) in a tail clip bleeding and FeCl3-induced thrombosis model. Respective tissue disposition of FIX was evaluated using immunofluorescence. RESULTS: In vitro characterization demonstrated comparable clotting activity of rFIX proteins. The PK profile showed that rFIXK5A displayed the highest plasma exposure compared to rFIXWT and rFIXK5R. Immunofluorescence evaluation of liver tissue showed that rFIXK5R was detectable up to 24 hours, whereas rFIXWT and rFIXK5A were detectable only up to 15 minutes. In the tail clip bleeding model, rFIXK5R displayed significant hemostatic protection against bleeding incidence for up to 72 hours postintravenous administration of 50 IU/kg, whereas the efficacy of rFIXK5A was already reduced at 24 hours. Similarly, in the mesenteric artery thrombus model, rFIXK5R and rFIXWT demonstrated prolonged efficacy compared to rFIXK5A. CONCLUSION: Using two different in vivo models of hemostasis and thrombosis, we demonstrate that mutated rFIX protein with enhanced binding (rFIXK5R) to extravascular space confers prolonged hemostatic efficacy in vivo despite lower plasma exposure, whereas rFIXK5A rapidly lost its efficacy despite higher plasma exposure.

Increased potency of recombinant VWF D'D3 albumin fusion proteins engineered for enhanced affinity for coagulation factor VIII.

BACKGROUND: We have recently reported on a recombinant von Willebrand factor (VWF) D'D3 albumin fusion protein (rD'D3-FP) developed to extend the half-life of coagulation factor VIII (FVIII) for the treatment of hemophilia A. Based on predictive modelling presented in this study, we hypothesized that modifying rD'D3-FP to improve FVIII interaction would reduce exchange with endogenous VWF and provide additional FVIII half-life benefit. OBJECTIVES: The aim of this study was to identify novel rD'D3-FP variants with enhanced therapeutic efficacy in extending FVIII half-life. METHODS: Through both directed mutagenesis and random mutagenesis using a novel mammalian display platform, we identified novel rD'D3-FP variants with increased affinity for FVIII (rVIII-SingleChain) under both neutral and acidic conditions and assessed their ability to extend FVIII half-life in vitro and in vivo. RESULTS: In rat preclinical studies, rD'D3-FP variants with increased affinity for FVIII displayed enhanced potency, with reduced dose levels required to achieve equivalent rVIII-SingleChain half-life extension. In cell-based imaging studies in vitro, we also demonstrated reduced dissociation of rVIII-SingleChain from the rD'D3-FP variants within acidic endosomes and more efficient co-recycling of the rD'D3-FP/rVIII-SingleChain complex via the FcRn recycling system. CONCLUSIONS: In summary, at potential clinical doses, the rD'D3-FP variants provide marked benefits with respect to dose levels and half-life extension of co-administered FVIII, supporting their development for use in the treatment of hemophilia A.

Evaluation of urinary biomarkers for early detection of acute kidney injury in a rat nephropathy model.

INTRODUCTION: The implementation of novel, reliable biomarkers for the early and differential diagnosis of acute kidney injury (AKI) could greatly improve the timely treatment and prevention of disease progression, particularly since the current gold standards for detecting kidney injury such as serum creatinine (SCr) and blood urea nitrogen (BUN) lack sensitivity and specificity. We evaluated novel urinary kidney injury biomarkers focusing on early detection and better prediction of AKI with higher sensitivity and specificity. METHODS: In the rat, urinary biomarkers for kidney injury, i.e. albumin, beta-2-microglobulin (B2M), clusterin, cystatin C, kidney injury molecule-1 (KIM-1), neutrophil gelatinase-associated lipocalin (NGAL), osteopontin (OPN), and total protein (TP), were investigated in an AKI model using different hyperosmolar and high-dose solutions, i.e. mannitol, sucrose, and contrast medium (CM), as acute single insults leading to kidney injury. Additionally, dose-dependency of sucrose was investigated and effects were compared to the sucrose- and iron-containing marketed drug Venofer®. RESULTS: Levels of excreted urinary biomarkers correlated with severity of AKI, exhibited a dose-dependent response to sucrose treatment, and demonstrated evidence of recovery from kidney injury with transient and reversible changes. The exceptions were KIM-1 and NGAL, which showed later responses following CM and iron-induced renal injury. All biomarkers outperformed plasma creatinine (PCr), BUN, and histopathology, with regard to practicability and/or detection of proximal tubular injury. DISCUSSION: The use of a panel of urinary kidney injury biomarkers emerged as an early, sensitive, and predictive tool to detect AKI showing enhanced sensitivity compared to current state-of-the-art markers.

FVIII half-life extension by coadministration of a D'D3 albumin fusion protein in mice, rabbits, rats, and monkeys.

A novel mechanism for extending the circulatory half-life of coagulation factor VIII (FVIII) has been established and evaluated preclinically. The FVIII binding domain of von Willebrand factor (D'D3) fused to human albumin (rD'D3-FP) dose dependently improved pharmacokinetics parameters of coadministered FVIII in all animal species tested, from mouse to cynomolgus monkey, after IV injection. At higher doses, the half-life of recombinant FVIII (rVIII-SingleChain) was calculated to be increased 2.6-fold to fivefold compared with rVIII-SingleChain administered alone in rats, rabbits, and cynomolgus monkeys, and it was increased 3.1-fold to 9.1-fold in mice. Sustained pharmacodynamics effects were observed (ie, activated partial thromboplastin time and thrombin generation measured ex vivo). No increased risk of thrombosis was observed with coadministration of rVIII-SingleChain and rD'D3-FP compared with rVIII-SingleChain alone. At concentrations beyond the anticipated therapeutic range, rD'D3-FP reduced the hemostatic efficacy of coadministered rVIII-SingleChain. This finding might be due to scavenging of activated FVIII by the excessive amount of rD'D3-FP which, in turn, might result in a reduced probability of the formation of the tenase complex. This observation underlines the importance of a fine-tuned balance between FVIII and its binding partner, von Willebrand factor, for hemostasis in general.

Reproducibility of NMR analysis of urine samples: impact of sample preparation, storage conditions, and animal health status.

INTRODUCTION: Spectroscopic analysis of urine samples from laboratory animals can be used to predict the efficacy and side effects of drugs. This employs methods combining (1)H NMR spectroscopy with quantification of biomarkers or with multivariate data analysis. The most critical steps in data evaluation are analytical reproducibility of NMR data (collection, storage, and processing) and the health status of the animals, which may influence urine pH and osmolarity. METHODS: We treated rats with a solvent, a diuretic, or a nephrotoxicant and collected urine samples. Samples were titrated to pH 3 to 9, or salt concentrations increased up to 20-fold. The effects of storage conditions and freeze-thaw cycles were monitored. Selected metabolites and multivariate data analysis were evaluated after (1)H NMR spectroscopy. RESULTS: We showed that variation of pH from 3 to 9 and increases in osmolarity up to 6-fold had no effect on the quantification of the metabolites or on multivariate data analysis. Storage led to changes after 14 days at 4°C or after 12 months at -20°C, independent of sample composition. Multiple freeze-thaw cycles did not affect data analysis. CONCLUSION: Reproducibility of NMR measurements is not dependent on sample composition under physiological or pathological conditions.

Safety pharmacology--current and emerging concepts.

Safety pharmacology (SP) is an essential part of the drug development process that aims to identify and predict adverse effects prior to clinical trials. SP studies are described in the International Conference on Harmonisation (ICH) S7A and S7B guidelines. The core battery and supplemental SP studies evaluate effects of a new chemical entity (NCE) at both anticipated therapeutic and supra-therapeutic exposures on major organ systems, including cardiovascular, central nervous, respiratory, renal and gastrointestinal. This review outlines the current practices and emerging concepts in SP studies including frontloading, parallel assessment of core battery studies, use of non-standard species, biomarkers, and combining toxicology and SP assessments. Integration of the newer approaches to routine SP studies may significantly enhance the scope of SP by refining and providing mechanistic insight to potential adverse effects associated with test compounds.

Discovery of olodaterol, a novel inhaled beta2-adrenoceptor agonist with a 24 h bronchodilatory efficacy.

Compound 4p was identified from a series of 6-hydroxy-4H-benzo[1,4]oxazin-3-ones as potent agonist of the human beta2-adrenoceptor with a high beta1/beta2-selectivity. A complete reversal of acetylcholine-induced bronchoconstriction which lasted over the whole study period of 5h was demonstrated for 4p in a guinea pig in vivo model without any signs of cardiovascular effects up to 10-fold above the first dose reaching 100% bronchoprotection. The enantiomerically pure (R)-form of 4p exerted a bronchodilatory efficacy over 24 h in dogs and guinea pigs in the absence of systemic pharmacodynamic effects. Formoterol which was tested as comparator in the same in vivo models of acetylcholine-induced bronchoconstriction did not retain efficacy after 24 h. In summary, the preclinical profile of compound (R)-4p (olodaterol, also known as BI 1744 CL) suggests a potential for once-daily dosing in man accompanied with an improved safety profile.

NMR-based urine analysis in rats: prediction of proximal tubule kidney toxicity and phospholipidosis.

INTRODUCTION: The aim of safety pharmacology is early detection of compound-induced side-effects. NMR-based urine analysis followed by multivariate data analysis (metabonomics) identifies efficiently differences between toxic and non-toxic compounds; but in most cases multiple administrations of the test compound are necessary. We tested the feasibility of detecting proximal tubule kidney toxicity and phospholipidosis with metabonomics techniques after single compound administration as an early safety pharmacology approach. METHODS: Rats were treated orally, intravenously, inhalatively or intraperitoneally with different test compounds. Urine was collected at 0-8 h and 8-24 h after compound administration, and (1)H NMR-patterns were recorded from the samples. Variation of post-processing and feature extraction methods led to different views on the data. Support Vector Machines were trained on these different data sets and then aggregated as experts in an Ensemble. Finally, validity was monitored with a cross-validation study using a training, validation, and test data set. RESULTS: Proximal tubule kidney toxicity could be predicted with reasonable total classification accuracy (85%), specificity (88%) and sensitivity (78%). In comparison to alternative histological studies, results were obtained quicker, compound need was reduced, and very importantly fewer animals were needed. In contrast, the induction of phospholipidosis by the test compounds could not be predicted using NMR-based urine analysis or the previously published biomarker PAG. DISCUSSION: NMR-based urine analysis was shown to effectively predict proximal tubule kidney toxicity after single compound administration in rats. Thus, this experimental design allows early detection of toxicity risks with relatively low amounts of compound in a reasonably short period of time.

Measurement of glomerular filtration rate in the conscious rat.

INTRODUCTION: Glomerular filtration rate (GFR) is an important parameter for studying drug-induced impairments on renal function in rats. The GFR is calculated from the concentration of creatinine and blood urea nitrogen (BUN) in serum and in urine, respectively. Following current protocols serum and urine samples must be taken from the same animal. Thus, in order to determine time-dependent effects it is necessary to use for each time point one separated group of animals. We developed a statistical test which allows analyzing the GFR from two different groups of animals: one used for repeated serum and the other one used for repeated urine analysis. METHODS: Serum and urine samples were taken from two different sets of rats which were otherwise treated identically, i.e. drug doses, routes of administration (per os or per inhalation) and tap water loading. For each dose group GFR mean, standard deviation and statistical analysis to identify differences between the dose groups were determined. RESULTS: After determination of the optimal time points for measurements, the effect on GFR of the three reference compounds, furosemide, hydrochlorothiazide and formoterol, was calculated. The results showed that the diuretic drugs furosemide and hydrochlorothiazide decreased the GFR and the antidiuretic drug formoterol increased the GFR, as counter regulation on urine loss or urine retention, respectively. DISCUSSION: A mathematical model and the corresponding algorithm were developed, which can be used to calculate the GFR, and to test for differences between groups from two separated sets of rats, one used for urine, and the other one for serum analysis. This new method has the potential to reduce the number of animals needed and to improve the quality of data generated from various groups of animals in renal function studies.

Effect of commonly used vehicles on gastrointestinal, renal, and liver function in rats.

INTRODUCTION: Solubility is often a limiting factor when testing new compounds in animal experiments. Various solubilizing agents may be used, but each have their own pharmacological effects. We investigated the effects of selected vehicles having different chemical characteristics on gastrointestinal, renal, and liver function. METHODS: Rats were treated orally, intravenously or intraperitoneally and gastric emptying, intestinal transit, renal, and liver function were investigated. RESULTS: Gastrointestinal motility was influenced by hydroxyethylcellulose, hydroxypropyl-beta-cyclodextrin (HPbetaCD), HPgammaCD, DMSO, polyethylene glycol 400 (PEG 400), fat emulsion, and the corresponding emulsifier. Liver function was affected by HPbetaCD, HPgammaCD, DMSO, PEG 400, Polysorbate 80, Cremophor RH 40, and fat emulsion. An increase in liver enzymes was observed after PEG 400 and Polysorbate 80. DMSO interfered with clinical chemistry measurements in serum. Urinary function was modified by HPgammaCD, DMSO, PEG 400, and Polysorbate 80, while enhanced urine enzyme excretion was observed after HPbetaCD, HPgammaCD, DMSO, PEG 400, and Polysorbate 80. DISCUSSION: Most of the investigated vehicles changed gastrointestinal, renal, and/or liver parameters after application of a certain threshold dose for each assay. No "best" vehicle could be identified that may be used in each test system. Thus, vehicles must be selected not only on their chemical characteristics but also on their potential pharmacological activity in a given test system.

Re-evaluation of thin layer chromatography as an alternative method for the quantification of prostaglandins from rat Kupffer cells.

In contrast to conventionally used immunoassays, thin layer chromatography (TLC)--by prelabeling of cells with radioactive arachidonic acid (AA)--allows to differentiate between cellularly built and added prostanoids and thus to investigate feedback effects of prostanoids on their own release. PGD2, TXB2 and PGE2 released from zymosan-stimulated Kupffer cells were separated with distinct RF-values, corresponding to those of the pure substances. Quantification of PGD2 and PGE2 gave comparable results with TLC and immunoassays, but measurement in the presence of added prostanoids was only possible with TLC. Moreover TLC was superior to immunoassays in having a longer linear range while being comparably sensitive. Cellularly built TXB2 in its radioactively labeled form was not detectable by TLC. Inhibition of TXB2 release by externally added AA or technical artifacts were excluded, suggesting that the cellular AA-pools used for prostaglandin and thromboxane synthesis differ in their accessibility for added AA. Thus, TLC is a simple, sensitive and precise method for the quantification of cellularly built prostaglandins but not of thromboxane even in the presence of added prostanoids.

Differences in the involvement of prostanoids from Kupffer cells in the mediation of anaphylatoxin C5a-, zymosan-, and lipopolysaccharide-dependent hepatic glucose output and flow reduction.

Various inflammatory stimuli such as anaphylatoxin C5a, zymosan, and lipopolysaccharides (LPSs) have been reported both to enhance glucose output in the perfused rat liver and to induce prostanoid (ie, prostaglandin and thromboxane) release from Kupffer cells, the resident liver macrophages. Because prostanoids can enhance glucose output from hepatocytes, it was the aim of this study to compare the possible roles of prostanoids released after C5a, zymosan, and LPS in the mediation of hepatic glucose output. In perfused livers both C5a and zymosan immediately enhanced glucose output, reduced flow, and induced prostanoid overflow into the hepatic vein, but with different quantities and kinetics. Only the C5a-induced but not the zymosan-induced effects were abrogated by inhibitors of prostanoid signaling as the prostanoid synthesis inhibitor indomethacin and the thromboxane receptor antagonist daltroban. In contrast to C5a and zymosan, LPS had no effect on glucose output, flow rate, or prostanoid overflow. In isolated Kupffer cells, C5a and zymosan induced maximal release of prostaglandins D(2) and E(2) and of thromboxane A(2) within a period of 0 to 2 minutes and 5 to 15 minutes, respectively. In pulse-chase experiments, maximal prostanoid release was already observed after 2 minutes of continuous stimulation with C5a, but only after 10 to 15 minutes of continuous stimulation with zymosan. LPS-dependent prostanoid release was not seen before 1 hour. Thus, even though C5a, zymosan, and LPS induced prostanoid release from Kupffer cells, only C5a quickly regulated hepatic glucose metabolism in a prostanoid-dependent manner (due to the kinetics and quantities of prostanoids released).

Inhibition of prostaglandin D2 clearance in rat hepatocytes by the thromboxane receptor antagonists daltroban and ifetroban and the thromboxane synthase inhibitor furegrelate.

Prostanoids, i.e. prostaglandins and thromboxane, regulate liver-specific functions both in homeostasis and during defense reactions. For example, prostanoids are released from Kupffer cells, the resident liver macrophages, in response to the inflammatory mediator anaphylatoxin C5a, and mediate an enhanced glucose output from hepatocytes as energy supply. In perfused rat livers, the thromboxane receptor antagonist daltroban enhanced C5a-induced prostanoid overflow and reduced glucose output. It was the aim of this study to elucidate whether daltroban interfered with prostanoid release from Kupffer cells or prostanoid clearance by hepatocytes, and/or whether it directly influenced prostanoid-dependent glucose metabolism in these cells. In perfused rat livers, daltroban enhanced prostaglandin (PG)D(2) overflow not only after infusion of C5a (15-fold), but also after PGD(2) (10-fold). Neither daltroban nor another receptor antagonist, ifetroban, or the thromboxane synthase inhibitor furegrelate enhanced prostanoid release from Kupffer cells. In contrast, all inhibitors reduced clearance, i.e. uptake and degradation, of PGD(2) by hepatocytes: within 5 min uptake of 1 nmol/L PGD(2) was reduced from 43+/-5 fmol (controls) to 22+/-6 fmol (daltroban), 24+/-6 fmol (ifetroban) and 21+/-6 fmol (furegrelate). PGD(2) in the medium was reduced to 39+/-7% in the controls, but remained at 93+/-9%, 93+/-11% and 60+/-3% in the presence of the inhibitors. PGD(2)-dependent glucose output in the perfused liver or activation of glycogen phosphorylase in isolated hepatocytes remained unaffected by daltroban. These data clearly demonstrate that the thromboxane-inhibitors reduced PGD(2) clearance by hepatocytes, presumably by inhibition of prostanoid transport into the cells. In contrast, they did not interfere with PGD(2)-dependent glucose metabolism, suggesting an independent mechanism for the inhibition of glucose output from the liver.

Inhibition by prostaglandin E(2) of anaphylatoxin C5a- but not zymosan-induced prostanoid release from rat Kupffer cells.

The proinflammatory anaphylatoxin C5a induces the release of prostanoids, ie, prostaglandins (PG) and thromboxane (TX), from the resident liver macrophages (Kupffer cells [KC]). Because KC themselves express prostanoid receptors, prostanoids--besides having paracrine functions--might regulate their own release in an autocrine loop. So far, such a possible feedback regulation has not been investigated systematically, probably because of methodological difficulties to measure newly synthesized prostanoids in the presence of added prostanoids. Here, after prelabeling of phospholipids with [(14)C]arachidonate, cellularly formed [(14)C]prostanoids were determined in the presence of added unlabelled prostanoids by thin layer chromatography. In cultured KC, recombinant rat C5a (rrC5a) rapidly increased PGD(2), PGE(2), and TXA(2) release, which was strongly reduced by PGE(2), but neither by PGD(2) nor by the TXA(2) analog U46619. The inhibitory effect of PGE(2) was mimicked by cAMP, indicating that the G(s)-coupled PGE(2) receptors type 2 or 4 were involved. Zymosan also enhanced prostanoid release from KC, but with slightly slower kinetics; this action was neither inhibited by PGE(2) nor by cAMP. Also in perfused rat livers, rrC5a enhanced prostanoid release from KC as shown by prostanoid overflow and thereby indirectly increased glucose output from hepatocytes. Again, PGE(2), but not PGD(2), inhibited rrC5a-elicited prostanoid overflow. This resulted in a complete inhibition of rrC5a-induced, prostanoid-mediated glucose output. Thus, PGE(2) can inhibit specifically the C5a-induced prostanoid release from KC via a feedback mechanism and thereby limit prostanoid-mediated hepatocellular defense reactions, eg, glucose release.

Expression of the serine/threonine kinase hSGK1 in chronic viral hepatitis.

The human serine/threonine kinase hSGK1 is expressed ubiquitously with highest transcript levels in pancreas and liver. This study has been performed to determine the hSGK1 distribution in normal liver and its putative role in fibrosing liver disease. HSGK1-localization was determined by in situ hybridization, regulation of hSGK1-transcription by Northern blotting, fibronectin synthesis and hSGK1 phosphorylation by Western blotting. In normal liver hSGK1 was mainly transcribed by Kupffer cells. In liver tissue from patients with chronic viral hepatitis, hSGK1 transcript levels were excessively high in numerous activated Kupffer cells and inflammatory cells localized within fibrous septum formations. HSGK1 transcripts were also detected in activated hepatic stellate cells. Accordingly, Western blotting revealed that tissue from fibrotic liver expresses excessive hSGK1 protein as compared to normal liver. TGF-beta1 (2 ng/ml) increases hSGK1 transcription in both human U937 macro-phages and HepG2 hepatoma cells. H(2)O(2) (0.3 mM) activated hSGK1 and increased fibronectin formation in HepG2 cells overexpressing hSGK1 but not in HepG2 cells expressing the inactive mutant hSGK1(K127R). In conclusion hSGK1 is upregulated by TGF-beta1 during hepatitis and may contribute to enhanced matrix formation during fibrosing liver disease.