LTA4H inhibitor LYS006: Clinical PK/PD and safety in a randomized phase I clinical trial.

LYS006 is a novel, highly potent and selective, new-generation leukotriene A4 hydrolase (LTA4H) inhibitor in clinical development for the treatment of neutrophil-driven inflammatory diseases. We describe the complex pharmacokinetic to pharmacodynamic (PD) relationship in blood, plasma, and skin of LYS006-treated nonclinical species and healthy human participants. In a randomized first in human study, participants were exposed to single ascending doses up to 100 mg and multiple ascending doses up to 80 mg b.i.d.. LYS006 showed rapid absorption, overall dose proportional plasma exposure and nonlinear blood to plasma distribution caused by saturable target binding. The compound efficiently inhibited LTB4 production in human blood and skin blister cells, leading to greater than 90% predose target inhibition from day 1 after treatment initiation at doses of 20 mg b.i.d. and above. Slow re-distribution from target expressing cells resulted in a long terminal half-life and a long-lasting PD effect in ex vivo stimulated blood and skin cells despite low plasma exposures. LYS006 was well-tolerated and demonstrated a favorable safety profile up to highest doses tested, without any dose-limiting toxicity. This supported further clinical development in phase II studies in predominantly neutrophil-driven inflammatory conditions, such as hidradenitis suppurativa, inflammatory acne, and ulcerative colitis.

Human Pharmacokinetics of LYS006, an Oral Leukotriene A4 Hydrolase Inhibitor Displaying Target-Mediated Drug Disposition.

LYS006 is a potent leukotriene A4 hydrolase inhibitor currently in clinical development for long-term treatment of various neutrophil-driven inflammatory conditions. Here, we present pharmacokinetics from the first-in-human study with complementary metabolism and transporter profiling data. The randomized first-in-human study included nine cohorts receiving 5-2*100 mg of LYS006 or placebo, a crossover food-effect part, and a multiple-dose part consisting of two fasted (5 mg and 15 mg once daily) and three fed cohorts (20-80 mg twice a day) of LYS006 or placebo. LYS006 and metabolites were assessed in plasma and urine, and transporters involved in LYS006 disposition were analyzed in vitro. Systemic plasma exposure increased with dose; steady-state exposure was dose proportional up to 40 mg twice a day. Steady state was achieved after ∼3 days, with mean accumulation of 2.1-fold for 5 mg once daily and ≤1.4-fold for all higher doses. Despite limited accumulation, a long terminal half-life (T1/2) was observed. The long T1/2 and saturable binding to blood cells, which causes a highly nonlinear blood-to-plasma distribution, reflect a strong impact of target binding on drug distribution at lower concentrations. Skin biopsy and blister fluid concentration data indicated saturable binding in the former but not the latter, suggesting saturable binding in tissues beyond blood. Major excretion of LYS006 (∼90% of dose) through urine at steady state triggered renal transporter investigations that identified LYS006 as a substrate of organic anion transporter (OAT)3, OAT4, breast cancer resistance protein, and multidrug resistance-associated protein 4. Seven metabolites were identified in human plasma and urine, comprising only 4% of the dose recovered in urine at steady state. SIGNIFICANCE STATEMENT: Pharmacokinetic data from a first-in-human study combined with in vitro work support dose and regimen selection for patient studies with LYS006 and provide guidance on drug interaction investigations and other clinical pharmacology work needed for further development. Mass balance information at steady state without the use of a radiolabel, skin concentrations, and identification of the major clearance pathway, as well as the transporters driving elimination, make this a particularly conclusive early study despite nonlinear pharmacokinetics impacted by target binding.

Clinical Investigation of Metabolic and Renal Clearance Pathways Contributing to the Elimination of Fevipiprant Using Probenecid as Perpetrator.

Fevipiprant, an oral, nonsteroidal, highly selective, reversible, and competitive prostaglandin D2 receptor 2 antagonist, is eliminated by glucuronidation and by direct renal excretion predominantly via organic anion transporter (OAT) 3. This study aimed to assess the effect of simultaneous UDP-glucuronosyltransferase (UGT) and OAT3 inhibition by probenecid on the pharmacokinetics of fevipiprant and its acyl glucuronide (AG) metabolite to support the dosing recommendation of fevipiprant in the presence of drugs inhibiting these pathways; however, phase III clinical trial results did not support its submission. This was a single-center, open-label, single-sequence, two-period crossover study in healthy subjects. Liquid chromatography with tandem mass spectrometry was used to measure concentrations of fevipiprant and its AG metabolite in plasma and urine. In the presence of probenecid, the mean maximum concentrations of fevipiprant increased approximately 1.7-fold, and the area under the concentration-time curve in plasma increased approximately 2.5-fold, whereas the mean apparent volume of distribution and the AG metabolite:fevipiprant ratio decreased. The apparent systemic clearance decreased by approximately 60% and the renal clearance decreased by approximately 88% in the presence of probenecid. Using these data and those from previous studies, the relative contribution of OAT and UGT inhibition to the overall effect of probenecid was estimated. Furthermore, a general disposition scheme for fevipiprant was developed, showing how a perpetrator drug such as probenecid, which interferes with two key elimination pathways of fevipiprant, causes only a moderate increase in exposure and allows estimation of the drug-drug inhibition when only one of the two pathways is inhibited. SIGNIFICANCE STATEMENT: In this drug-drug interaction (DDI) study, probenecid was used as a tool to inhibit both glucuronidation and active renal secretion of fevipiprant. The combination of plasma and urine pharmacokinetic data from this study with available data allowed the development of a quantitative scheme to describe the fate of fevipiprant in the body, illustrating why the DDI effect on fevipiprant is weak-to-moderate even if a perpetrator drug inhibits several elimination pathways.

A Study of the Effect of Cyclosporine on Fevipiprant Pharmacokinetics and its Absolute Bioavailability Using an Intravenous Microdose Approach.

This drug-drug interaction study determined the effect of cyclosporine, an inhibitor of organic anion transporting polypeptide (OATP) 1B3 and P-gp, on the pharmacokinetics (PK) of fevipiprant, an oral, highly selective, competitive antagonist of the prostaglandin D2 receptor 2 and a substrate of the two transporters. The concomitant administration of an intravenous microdose of stable isotope-labeled fevipiprant provided the absolute bioavailability of fevipiprant as well as mechanistic insights into its PK and sensitivity to drug interactions. Liquid chromatography-mass spectrometry/mass spectrometry was used to measure plasma and urine concentrations. Geometric mean ratios [90% confidence interval (CI)] for oral fevipiprant with or without cyclosporine were 3.02 (2.38, 3.82) for C max, 2.50 (2.17, 2.88) for AUClast, and 2.35 (1.99, 2.77) for AUCinf The geometric mean ratios (90% CI) for fevipiprant intravenous microdose with or without cyclosporine were 1.04 (0.86, 1.25) for C max, 2.04 (1.83, 2.28) for AUClast, and 1.95 (1.76, 2.16) for AUCinf The absolute bioavailability for fevipiprant was approximately 0.3 to 0.4 in the absence and 0.5 in the presence of cyclosporine. The intravenous microdose allowed differentiation between systemic and presystemic effects of cyclosporine on fevipiprant, demonstrating a small (approximately 1.2-fold) presystemic effect of cyclosporine and a larger (approximately twofold) effect on systemic elimination of fevipiprant. Uptake by OATP1B3 appears to be the rate-limiting step in the hepatic elimination of fevipiprant, whereas P-gp does not have a relevant effect on oral absorption. SIGNIFICANCE STATEMENT: The drug interaction investigated here with cyclosporine, an inhibitor of several drug transporters, provides a refined quantitative understanding of the role of active transport processes in liver and intestine for the absorption and elimination of fevipiprant as well as the basis to assess the need for dose adjustment in the presence of transporter inhibitors. The applied intravenous microdose approach presents a strategy to maximize learnings from a trial, limit the number and duration of clinical trials, and enhance mechanistic drug-drug interaction understanding.

Absorption, Distribution, Metabolism, and Excretion of the Oral Prostaglandin D2 Receptor 2 Antagonist Fevipiprant (QAW039) in Healthy Volunteers and In Vitro.

Fevipiprant is a novel oral prostaglandin D2 receptor 2 (DP2; also known as CRTh2) antagonist, which is currently in development for the treatment of severe asthma and atopic dermatitis. We investigated the absorption, distribution, metabolism, and excretion properties of fevipiprant in healthy subjects after a single 200-mg oral dose of [14C]-radiolabeled fevipiprant. Fevipiprant and metabolites were analyzed by liquid chromatography coupled to tandem mass spectrometry and radioactivity measurements, and mechanistic in vitro studies were performed to investigate clearance pathways and covalent plasma protein binding. Biotransformation of fevipiprant involved predominantly an inactive acyl glucuronide (AG) metabolite, which was detected in plasma and excreta, representing 28% of excreted drug-related material. The AG metabolite was found to covalently bind to human plasma proteins, likely albumin; however, in vitro covalent binding to liver protein was negligible. Excretion was predominantly as unchanged fevipiprant in urine and feces, indicating clearance by renal and possibly biliary excretion. Fevipiprant was found to be a substrate of transporters organic anion transporter 3 (OAT3; renal uptake), multidrug resistance gene 1 (MDR1; possible biliary excretion), and organic anion-transporting polypeptide 1B3 (OATP1B3; hepatic uptake). Elimination of fevipiprant occurs via glucuronidation by several uridine 5'-diphospho glucuronosyltransferase (UGT) enzymes as well as direct excretion. These parallel elimination pathways result in a low risk of major drug-drug interactions or pharmacogenetic/ethnic variability for this compound.

Equilibrium gel filtration to measure plasma protein binding of very highly bound drugs.

For very highly bound drugs (fu < 2%), the determination of the unbound fraction in plasma (fu) and a reliable estimation of protein-binding differences across species, populations, or concentrations is challenging. The difficulty is not mostly assay sensitivity but rather experimental bias. In equilibrium gel filtration (EGF)--opposite to the commonly used methods--the amount bound at a set-free concentration is determined. Therefore, signals and differences are bigger for more highly protein-bound drugs. We describe here a new experimental set-up developed to investigate binding in plasma and compare results with those obtained with standard methods for nine Novartis compounds. The method was then applied for two drugs for which it was challenging to obtain precise data with standard methods: midostaurin and siponimod. Despite the very high binding (fu ≤ 0.1%), precise estimation of up to 10-fold species differences relevant for safety assessments was possible. Evidence for the correctness of the data by comparison with other pharmacokinetics parameters is provided. Sensitivity to potential sources of experimental bias is compared with standard methods and advantages and disadvantages of the methods are discussed. In conclusion, EGF allows accurate determination of fu for very highly bound drugs and differentiation even above 99.9% of binding.

Metabolism and disposition of the oral absorption enhancer 14C-radiolabeled 8-(N-2-hydroxy-5-chlorobenzoyl)-amino-caprylic acid (5-CNAC) in healthy postmenopausal women and supplementary investigations in vitro.

8-(N-2-hydroxy-5-chlorobenzoyl)-amino-caprylic acid (5-CNAC), a compound lacking pharmacological activity enhances the absorption of salmon calcitonin, when co-administered. Disposition and biotransformation of 5-CNAC was studied in six healthy postmenopausal women following a single oral dose of 200mg (14)C-radiolabeled 5-CNAC (as disodium monohydrate salt). Blood, plasma, urine and feces collected over 7 days were analyzed for radioactivity. Metabolite profiles were determined in plasma and excreta and metabolite structures were elucidated by LC-MS/MS, LC-(1)H NMR, enzymatic methods and by comparison with reference compounds. Oral 5-CNAC was safe and well tolerated in this study population. 5-CNAC absorption was rapid (t(max)=0.5h; C(max)=9.00 ± 2.74 µM (mean ± SD, n=6) and almost complete. The elimination half-life (t(½)) was 1.5 ± 1.1h. The radioactive dose was excreted mainly in urine (≥ 90%) in form of metabolites and 0.071% as intact 5-CNAC. Excretion of radioactivity in feces was minor and mostly as metabolites (<3%). Radioactivity in plasma reached C(max) (35.4 ± 7.9 µM) at 0.75 h and declined with a half-life of 13.9 ± 4.3h. 5-CNAC accounted for 5.8% of the plasma radioactivity AUC(0-24h). 5-CNAC was rapidly cleared from the systemic circulation, primarily by metabolism. Biotransformation of 5-CNAC involved: (a) stepwise degradation of the octanoic acid side chain and (b) conjugation of 5-CNAC and metabolites with glucuronic acid at the 2-phenolic hydroxyl group. The metabolism of 5-CNAC in vivo could be reproduced in vitro in human hepatocytes. No metabolism of 5-CNAC was observed in human liver microsomes.

Biodistribution and plasma protein binding of zoledronic acid.

The bisphosphonate zoledronic acid is a potent inhibitor of osteoclast-mediated bone resorption. To investigate drug biodistribution and elimination, (14)C-zoledronic acid was administered intravenously to rats and dogs in single or multiple doses and assessed for its in vitro blood distribution and plasma protein binding in rat, dog, and human. Drug exposure in plasma, bones, and noncalcified tissues was investigated up to 240 days in rats and 96 h in dogs using radiometry after dissection. Drug biodistribution in the rat and within selected bones from dog was assessed by autoradiography. Concentrations of radioactivity showed a rapid decline in plasma and noncalcified tissue but only a slow decline in bone, to approximately 50% of peak at 240 days post dose, whereas the terminal half-lives (50-200 days) were similar in bone and noncalcified tissues, suggesting redistribution of drug from the former rather than prolonged retention in the latter. Uptake was highest in cancellous bone and axial skeleton. At 96 h after dose, the fraction of dose excreted was 36% in rat and 60% in dog; 94 to 96% of the excreted radioactivity was found in urine. Blood/plasma concentration ratios were 0.52 to 0.59, and plasma protein binding of zoledronic acid was moderate to low in all species. The results suggest that a fraction of zoledronic acid is reversibly taken up by the skeleton, the elimination of drug is mainly by renal excretion, and the disposition in blood and noncalcified tissue is governed by extensive uptake into and slow release from bone.

Binding of pimecrolimus and tacrolimus to skin and plasma proteins: implications for systemic exposure after topical application.

Pimecrolimus and tacrolimus are calcineurin inhibitors used for the topical treatment of atopic dermatitis. Although structurally similar, they display specific differences including higher lipophilicity and lower skin permeation of pimecrolimus. The aim of the present study was to understand the reason for the differences in skin permeation; in addition, plasma protein binding of the two drugs was analyzed side by side as a basis for comparison of systemic exposure to free drug. Permeation of pimecrolimus and tacrolimus through a silicon membrane was found to be similar; therefore, we assumed that differences in skin permeation could be caused by differences in affinity to skin components. To test this hypothesis, we investigated binding of pimecrolimus and tacrolimus to a preparation of soluble human skin proteins. One binding protein of approximately 15 kDa, probably corresponding to macrophilin12, displayed a similar binding capacity for pimecrolimus and tacrolimus. However, less specific, nonsaturating binding to other proteins was approximately 3-fold higher for pimecrolimus. Because of the high local drug concentration after topical administration, the unspecific, high-capacity binding is probably dominating the permeation through skin. In plasma both drugs bound predominantly to lipoproteins, which may affect disposition differently from albumin binding. The unbound fraction of pimecrolimus in human plasma was approximately 9-fold lower compared with that of tacrolimus (0.4 +/- 0.1 versus 3.7 +/- 0.8%). In conclusion, these results provide an explanation for the observed lower systemic exposure to pimecrolimus than to tacrolimus after topical application and suggest that differences in systemic exposure to free drug might be even more pronounced.

ADME investigations of unnatural peptides: distribution of a 14C-labeled beta 3-octaarginine in rats.

The highly positively charged, cell-penetrating beta3-octaarginine has been prepared with a radioactive label by acetylation at the N-terminus with a doubly (14)C-labeled acetyl group ((14)CH3-(14)CO). With the radioactive compound, an ADME study (Absorption, Distribution, Metabolism, Excretion) was performed in male rats following an intravenous or oral dose of 1 mg/kg. Sampling was carried out after periods ranging from 5 min to 4 d or 7 d for blood/excretia and quantitative whole-body autoradioluminography (QWBA), respectively. After p.o. dosing, no systemic exposure to peptide-related radioactivity was observed, and the dose was completely excreted in the feces within 24 h suggesting the absence of relevant absorption; less than 3% of the i.v. dose was excreted from the animals within 4 d. Blood levels, after i.v. dosing, dropped within 4 d to less than 2% of Cmax and decreased afterwards only very slowly. No metabolites were observed in the systemic circulation. QWBA Data indicated that the distribution of the acetyl-beta-octaarginine-related radioactivity in the organs and tissues shifted over time. Notably, after 7 d, the highest concentration was measured in the lymph nodes, and the largest amount was found in the liver. A comparison with the results of two previous ADME investigations of beta-peptides (cf. Table 1) reveals that the distribution of the compounds within the animals is structure-dependent, and that there is a full range from oral availability with rather rapid excretion (of a tetrapeptide) to essentially complete lack of both oral absorption and excretion after i.v. administration (of a highly charged octapeptide). A discussion is presented about the in vivo stability and 'drug-ability' of peptides. In general, beta-peptides bearing proteinogenic side chains are compared with peptides consisting entirely of D-alpha-amino acid residues (the enantiomers of the 'natural' building blocks), and suggestions are made regarding a possible focus of future biomedical investigations with beta-peptides.

In vitro blood distribution and plasma protein binding of the iron chelator deferasirox (ICL670) and its iron complex Fe-[ICL670]2 for rat, marmoset, rabbit, mouse, dog, and human.

Deferasirox (Exjade, ICL670) is an orally active iron chelator. Two molecules of deferasirox can form a complex with ferric iron (Fe-[ICL670]2) that can be excreted, reducing body iron overload. The blood binding parameters across species and the interaction with human serum albumin were analyzed for deferasirox and its iron complex. Both molecules were very highly bound to plasma proteins in all the tested species with unbound fractions in plasma in the range of 0.4 to 1.8% and 0.2 to 1.2% for deferasirox and Fe-[ICL670]2, respectively; binding of the iron complex was either similar or higher in all the species. The high plasma protein binding was in line with a distribution mainly into the plasma fraction of blood; the fraction in plasma was around 100% for Fe-[ICL670]2 in all the species and 65 to 95% for deferasirox depending on the species. Investigations with isolated proteins pointed to serum albumin as the principal binding protein for deferasirox and its iron complex in human plasma. Competition binding experiments indicated that deferasirox at high concentrations displaced markers from the two main drug binding sites of human albumin, whereas Fe-[ICL670]2 displaced only warfarin. In the context of the pharmacokinetic properties of deferasirox and Fe-[ICL670]2, the data indicate the importance of plasma protein binding for their disposition and support a comparison of the pharmacokinetics of deferasirox and its iron complex across species. The low likelihood of clinically relevant drug displacement by deferasirox in plasma is discussed.

Exploring the antibacterial and hemolytic activity of shorter- and longer-chain beta-, alpha,beta-, and gamma-peptides, and of beta-peptides from beta2-3-aza- and beta3-2-methylidene-amino acids bearing proteinogenic side chains--a survey.

The antibacterial activities of 31 different beta-, mixed alpha/beta-, and gamma-peptides, as well as of beta-peptides derived from beta2-3-aza- and beta3-2-methylidene-amino acids were assayed against six pathogens (Enterococcus faecalis, Staphylococcus aureus, Streptococcus pneumoniae, Escherichia coli, Klebsiella pneumoniae, and Pseudomonas aeruginosa), and the results were compared with literature data. The interaction of these peptides with mammalian cells, as modeled by measuring the hemolysis of human erythrocytes, was also investigated. In addition to those peptides designed to fold into amphiphilic helical conformations with positive charges on one face of the helix, one new peptide with hemolytic activity was detected within the sample set. Moreover, it was demonstrated that neither cationic peptides used for membrane translocation (beta3-oligoarginines), nor mixed alpha/beta- or gamma-peptides with somatostatin-mimicking activities display unwanted hemolytic activity.

Hepatic transport of PKI166, an epidermal growth factor receptor kinase inhibitor of the pyrrolo-pyrimidine class, and its main metabolite, ACU154.

PKI166, a specific inhibitor of the tyrosine kinase activity of two epidermal growth factor receptors, was under development for the treatment of cancer. In preclinical studies PKI166 was mainly cleared by metabolism, and its metabolites were eliminated by biliary excretion, emphasizing the role of liver transport processes for its disposition. Here the transport properties of [14C]PKI166 and its main metabolite [14C]ACU154, an O-glucuronide, were analyzed using 1) Madin-Darby canine kidney II (MDCKII) cells stably transfected with human multidrug resistance-associated protein 2 (MRP2) and/or human organic anion-transporting peptide 2 (OATP2) and 2) liver canalicular membrane vesicles (CMVs) prepared from Wistar and mrp2-deficient TR- rats. Analysis of transport through MDCKII cells revealed that [14C]ACU154 was a substrate of MRP2 and OATP2. Rat mrp2 was shown to transport [14C]ACU154 with a Km of approximately 1 microM. [14C]PKI166 efficiently crossed MDCKII cells, particularly toward the apical side, but expression of MRP2 and/or OATP2 did not increase the flux. The effect of PKI166 and ACU154 on transport of [3H]estradiol-17beta-d-glucuronide (EG; via mrp2/MRP2 and OATP2) or [3H]taurocholic acid (TCA; via bile salt export pump (bsep) was analyzed. PKI166 inhibited the transport of [3H]EG by OATP2. ACU154 did strongly inhibit [3H]TCA uptake into CMVs from Wistar but not from TR- rats, demonstrating a dependence of bsep inhibition on mrp2 activity. ATP-dependent uptake of [3H]EG into CMVs from Wistar rats was inhibited by ACU154 but up to 4-fold increased by PKI166. In conclusion, OATP2 and MRP2/mrp2 were identified as transporters involved in ACU154 transport into bile. Both PKI166 and its O-glucuronide ACU154 affected mrp2/MRP2-, OATP2-, and/or bsep-mediated transport processes.

In vitro blood distribution and plasma protein binding of the tyrosine kinase inhibitor imatinib and its active metabolite, CGP74588, in rat, mouse, dog, monkey, healthy humans and patients with acute lymphatic leukaemia.

AIMS: To determine blood binding parameters of imatinib and its metabolite CGP74588 in humans and non-human species. METHODS: The blood distribution and protein binding of imatinib and CGP74588 were determined in vitro using (14)C labelled compounds. RESULTS: The mean fraction of imatinib in plasma (f(p)) was 45% in dog, 50% in mouse, 65% in rat, 70% in healthy humans and up to 92% in acute lymphatic leukaemia (AML) patients. Similarly, f(p) for CGP74588 was low in dog and monkey (30%), higher in rat, mouse and humans (70%) and highest in some AML patients (90%). The unbound fraction of imatinib and CGP74588 in plasma was lower in rat, mouse, healthy humans and AML patients (2.3-6.5% at concentrations < or = 5000 ng ml(-1)) compared to monkey and dog (7.6-19%). Both compounds displayed high binding to human alpha(1)-acid glycoprotein. AML patients had a reduced haematocrit and showed greatest variability in their blood binding parameters. CONCLUSION: Imatinib and CGP74588 displayed very similar blood binding parameters within all species/groups investigated. The five species clustered into two distinct groups with rat, mouse and humans being clearly different from dog and monkey. For both compounds, higher protein binding was associated with a decreased partitioning into blood cells.

Purification and characterization of the human adenosine A(2a) receptor functionally expressed in Escherichia coli.

The adenosine A(2a) receptor belongs to the seven transmembrane helix G-protein-coupled receptor family, is abundant in striatum, vasculature and platelets and is involved in several physiological processes such as blood pressure regulation and protection of cells during anoxia. For structural and biophysical studies we have expressed the human adenosine A(2a) receptor (hA2aR) at high levels inserted into the Escherichia coli inner membrane, and established a purification scheme. Expression was in fusion with the periplasmic maltose-binding protein to levels of 10-20 nmol of receptor per L of culture, as detected with the specific antagonist ligand [(3)H]ZM241385. As the receptor C-terminus was proteolyzed upon solubilization, a protease-resistant but still functional receptor was created by truncation to Ala316. Addition of the sterol, cholesteryl hemisuccinate, allowed a stable preparation of functional hA2aR solubilized in dodecylmaltoside to be obtained, and, increased the stability of the receptor solubilized in other alkylmaltosides. Purification to homogeneity was achieved in three steps, including ligand affinity chromatography based on the antagonist xanthine amine congener. The purified hA2aR fusion protein bound [(3)H]ZM241385 with a K(d) of 0.19 nm and an average B(max) of 13.7 nmol x mg(-1) that suggests 100% functionality. Agonist affinities for the purified solubilized receptor were higher than those for the membrane-bound form. Sufficient pure, functional hA2aR can now be prepared regularly for structural studies.