Downregulation of CYP1A2, CYP2B6, and CYP3A4 in Human Hepatocytes by Prolyl Hydroxylase Domain 2 Inhibitors via Hypoxia-Inducible Factor-α Stabilization.

Hypoxia-inducible factor (HIF) is associated with the expression of CYP, but the underlying mechanism remains uncertain. In this study, we investigated the effect of HIF-α stabilization caused by novel prolyl hydroxylase domain (PHD) 2 inhibitors, which are HIF-α stabilizers that mimic hypoxia, on the expressions of CYP1A2, CYP2B6, and CYP3A4 in human hepatocytes. An mRNA expression analysis of human hepatocytes treated with PHD2 inhibitors for 72 hours showed the downregulation of genes encoding CYP1A2, CYP2B6, and CYP3A4. The mRNA repressions were accompanied with an increase in erythropoietin protein, a marker of HIF-α stabilization, indicating that HIF-α stabilization was involved in the downregulation of the CYP isoforms. To understand the underlying mechanisms, we assessed the relationship between the expressions of the CYP isoforms and those of their regulating transcription factors [aryl hydrocarbon receptor (AhR), AhR nuclear translocator (ARNT), constitutive androstane receptor (CAR), pregnane X receptor (PXR), and retinoid X receptor (RXR)] in human hepatocytes treated with the HIF-α stabilizers. As a result, the mRNA level of AhR did not decrease, although ARNT expression was repressed. On the other hand, the mRNA expression levels of CAR, PXR, and RXR were repressed and closely associated with those of CYP2B6 and CYP3A4. Although the underlying mechanism of the downregulation for CYP1A2 remains unclear, the presently reported results suggest that the downregulation of CYP2B6 and CYP3A4 via HIF-α stabilization is caused by a decrease in the expressions of CAR, PXR, and RXR. SIGNIFICANCE STATEMENT: We showed that hypoxia-inducible factor (HIF)-α stabilization downregulates CYP1A2, CYP2B6, and CYP3A4 using prolyl hydroxylase domain 2 inhibitors, which are HIF-α stabilizers, as a new tool to mimic hypoxia in human hepatocytes. To understand the underlying mechanisms, we assessed the relationship between the expressions of the CYP isoforms and those of their regulating transcription factors. Our findings would contribute to a better understanding of the hypoxia-triggered regulatory mechanism of drug-metabolizing enzymes in human hepatocytes.

First-in-man-proof of concept study with molidustat: a novel selective oral HIF-prolyl hydroxylase inhibitor for the treatment of renal anaemia.

AIMS: Insufficient erythropoietin (EPO) synthesis is a relevant cause of renal anaemia in patients with chronic kidney disease. Molidustat, a selective hypoxia-inducible factor prolyl hydroxylase (HIF-PH) inhibitor, increases endogenous EPO levels dose dependently in preclinical models. We examined the pharmacokinetics, safety, tolerability and effect on EPO levels of single oral doses of molidustat in healthy male volunteers. METHODS: This was a single-centre, randomized, single-blind, placebo-controlled, group-comparison, dose-escalation study. Molidustat was administered at doses of 5, 12.5, 25, 37.5 or 50 mg as a polyethylene glycol-based solution. RESULTS: In total, 45 volunteers received molidustat and 14 received placebo. Molidustat was absorbed rapidly, and the mean maximum plasma concentration and area under the concentration-time curve increased dose dependently. The mean terminal half-life was 4.64-10.40 h. A significant increase in endogenous EPO was observed following single oral doses of molidustat of 12.5 mg and above. Geometric mean peak EPO levels were 14.8 IU l-1 (90% confidence interval 13.0, 16.9) for volunteers who received placebo and 39.8 IU l-1 (90% confidence interval: 29.4, 53.8) for those who received molidustat 50 mg. The time course of EPO levels resembled the normal diurnal variation in EPO. Maximum EPO levels were observed approximately 12 h postdose and returned to baseline after approximately 24-48 h. All doses of molidustat were well tolerated and there were no significant changes in vital signs or laboratory safety parameters. CONCLUSIONS: Oral administration of molidustat to healthy volunteers elicited a dose-dependent increase in endogenous EPO. These results support the ongoing development of molidustat as a potential new treatment for patients with renal anaemia.

Novel complex crystal structure of prolyl hydroxylase domain-containing protein 2 (PHD2): 2,8-Diazaspiro[4.5]decan-1-ones as potent, orally bioavailable PHD2 inhibitors.

We have discovered a novel complex crystal structure of the PHD2 enzyme with its inhibitor, the 2,8-diazaspiro[4.5]decan-1-one analogue 4b. The widely reported salt bridge between Arg383 of the enzyme and its inhibitors in all complex structures published thus far was not observed in our case. In our complex structure compound 4b forms several novel interactions with the enzyme, which include a hydrogen bond with Arg322, a π-cation interaction with Arg322, a π-π stacking with Trp389, and a π-π stacking with His313. Guided by the structural information, SAR studies were performed on the 2,8-diazaspiro[4.5]decan-1-one series leading to the discovery of compound 9p with high potency and good oral pharmacokinetic profile in mice.

Pharmacokinetics of Daprodustat and Metabolites in Individuals with Normal and Impaired Hepatic Function.

Daprodustat is a hypoxia-inducible factor-prolyl hydroxylase inhibitor in development for treatment of anemia of chronic kidney disease. We evaluated the role of hepatic impairment on daprodustat pharmacokinetics, pharmacodynamics, and tolerability. Participants with mild (Child-Pugh Class A, score 5-6) and moderate (Child-Pugh Class B, score 7-9) hepatic impairment and matched healthy controls were administered single 6-mg doses of daprodustat. Exposure parameters were determined for daprodustat and its six metabolites. Comparisons resulted in 1.5- and 2.0-fold higher daprodustat Cmax and area under the curve (AUC) exposures in participants with mild and moderate hepatic impairment, respectively, versus controls; Cmax in mild hepatic impairment was comparable to controls. Similarly, aligned with parent drug, unbound daprodustat Cmax and AUC exposures increased 1.6- to 2.3-fold in hepatic-impaired participants versus controls, and metabolite exposures were 1.2- to 2.0-fold higher in participants with hepatic impairment. Erythropoeitin (EPO) baseline-corrected AUC exposures were between 0.3-fold lower and 2.2-fold higher in matched controls versus hepatic-impaired participants. No serious or study drug-related adverse events were reported. Daprodustat exposure was increased in participants with moderate and mild hepatic impairment compared with matched controls; however, no meaningful differences in EPO were observed and no new safety concerns were identified (ClinicalTrials.gov: NCT03223337).

A validated method for the quantification of IOX-2, a potent prolyl hydroxylase inhibitor in equine urine and plasma using liquid chromatography-high-resolution mass spectrometry.

IOX-2 is a potent inhibitor of enzyme prolyl hydroxylases-2 (PHD) that plays a critical role in regulating hypoxia inducible factor (HIF) abundance and oxygen homeostasis. Federation for Equestrian Sports has listed HIF activators as prohibited substances to prevent their usage in doping. Consequently, it became essential to develop adequate knowledge and testing methods to detect it in equine sports drug testing samples. The validated method utilizes ultra-high-performance liquid chromatography coupled to high-resolution mass spectrometry in order to detect extremely low concentration of the analyte present in both matrices. Confirmation for the presence of the analyte was achieved by comparing ion ratios, retention time, and accurate mass. Method linear range for plasma was between 0.25 to 100 ng/ml, and limit of detection (LOD) was 0.075 ng/ml. The linear range for urine was 0.125 to 100 ng/ml, and LOD was 0.025 ng/ml. Intraday precision at 0.5, 10, and 50 ng/ml was between 4.0% and 9.7% for plasma and 4.2% and 10.4% for urine. Accuracy at 0.5, 10, and 50 ng/ml was between 91% and 94% for plasma and 99% and 103% for urine. Elimination profile of IOX-2 in equine plasma and urine was carried out using the developed method in which two horses were intravenously administered IOX-2 and samples were collected. Metabolic profile in plasma and urine was investigated. IOX-2 was detected for a minimum of 54 and 151 h of post administration in plasma and urine, respectively, thereby providing a valuable tool for evaluating its misuse in equine racing.

Effect of Kidney Function and Dialysis on the Pharmacokinetics and Pharmacodynamics of Roxadustat, an Oral Hypoxia-Inducible Factor Prolyl Hydroxylase Inhibitor.

BACKGROUND AND OBJECTIVES: Roxadustat is an orally active hypoxia-inducible factor prolyl hydroxylase inhibitor for anemia in chronic kidney disease. The pharmacokinetics, metabolic profile, and pharmacodynamics of roxadustat were investigated in subjects with different degrees of kidney function. METHODS: This phase 1 open-label study enrolled subjects with normal and severely impaired kidney function, and end-stage renal disease (ESRD) on continuous ambulatory peritoneal dialysis (CAPD) or automated peritoneal dialysis (APD) or hemodialysis/hemodiafiltration (HD/HDF). All subjects received a single 100-mg dose of oral roxadustat. Within a single-sequence, two-treatment period design (P1/P2), subjects with ESRD on HD/HDF received roxadustat 2 h after (P1) and 2 h before (P2) a dialysis session. Area under the plasma concentration-time curve (AUC) from administration to infinity (AUCinf), maximum concentration (Cmax), and terminal elimination half-life (t1/2) were assessed for roxadustat; AUC and Cmax were assessed for erythropoietin. RESULTS: Thirty-four subjects were enrolled and received roxadustat (normal kidney function, n = 12; severely impaired kidney function, n = 9; ESRD on CAPD/APD, n = 1; ESRD on HD/HDF, n = 12). The geometric least-square mean ratio of AUCinf was 223% and 195% in subjects with severely impaired kidney function and ESRD on HD/HDF, respectively, relative to subjects with normal kidney function; Cmax and t1/2 were comparable. The pharmacokinetic profile of roxadustat was not affected by HD/HDF. AUCinf and t1/2 for the metabolites of roxadustat increased in subjects with kidney impairment. The AUC and Cmax of erythropoietin increased in subjects with severely impaired kidney function or ESRD on HD/HDF. Roxadustat was well tolerated. CONCLUSIONS: Kidney function impairment increased the AUC of roxadustat and its metabolites. The Cmax and t1/2 of roxadustat were comparable among groups. Roxadustat and its metabolites were not cleared by HD/HDF. Clinical Trials Registration Number: NCT02965040.

Effect of Moderate Hepatic Impairment on the Pharmacokinetics of Vadadustat, an Oral Hypoxia-Inducible Factor Prolyl Hydroxylase Inhibitor.

Vadadustat is a hypoxia-inducible factor prolyl hydroxylase inhibitor in development for the treatment of anemia of chronic kidney disease. This phase 1, open-label, parallel-group, single-dose study evaluated the pharmacokinetics of 450-mg vadadustat in adults with moderate hepatic impairment (Child-Pugh class B) vs those with normal hepatic function. Primary end points were area under the plasma concentration-time curve (AUC) from dosing to last concentration and to infinity, as well as maximum concentration (Cmax ); additional pharmacokinetic parameters included time to Cmax (Tmax ) and half-life. Safety and tolerability were also assessed. All enrolled participants (n = 16) completed the study. Demographics were similar in both groups (overall, 100% White; 62.5% female; mean age, 59.2 years). Vadadustat plasma exposure was higher in the moderate hepatic impairment group, whereas maximum concentration was similar between groups. Point estimates of the hepatic impairment : normal geometric mean ratios (90% confidence interval) for AUC from dosing to last concentration, AUC from dosing to infinity, and Cmax were 1.05 (0.82-1.35), 1.06 (0.82-1.36), and 1.02 (0.79-1.32), respectively. Mean elimination half-life was 5.8 and 7.8 hours in the normal and hepatic impairment groups, respectively. Treatment-emergent adverse events were mostly mild in severity, and vadadustat was generally well tolerated. In conclusion, moderate hepatic impairment did not significantly impact vadadustat systemic exposure, and mild hepatic impairment is unlikely to alter vadadustat exposure.

A Mass Balance Study of 14 C-Labeled JTZ-951 (Enarodustat), a Novel Orally Available Erythropoiesis-Stimulating Agent, in Patients With End-Stage Renal Disease on Hemodialysis.

The mass balance, pharmacokinetics, and biotransformation of JTZ-951 (enarodustat), a novel hypoxia-inducible factor prolyl hydroxylase inhibitor, were characterized in patients (N = 6) with end-stage renal disease on hemodialysis. Following a 10-mg (100 µCi) oral dose of 14 C-JTZ-951, whole blood, feces, dialysate, and, if feasible, urine were obtained for pharmacokinetic assessments and for metabolite profiling and identification in appropriate matrices. Fecal excretion was the major route of elimination of radioactivity, and urinary excretion a minor route, with mean (coefficient of variation [%CV]) recovery of 77.1 (16.2)% and 10.9 (92.0)% of the dose, respectively. Radioactivity was not detected in the dialysate, and mean (%CV) total recovery in excreta was 88.0 (14.9)%. For parent JTZ-951 in plasma, the mean (%CV) effective half-life was 8.96 (7.7)% hours, and area under the curve over 24 hours comprised the majority (>80%) of total exposure, with relatively low variability in these pharmacokinetic variables. Based on profiling of plasma radioactivity, parent JTZ-951 was the predominant circulating component, accounting for 93.7% or more of radioactivity, and metabolite M2 (hydroxylated product) was the only detectable metabolite, but its exposure was minor (<5%) versus unchanged JTZ-951. In urine and feces, the predominant analyte was JTZ-951, and metabolite M2 was the predominant albeit minor metabolite, with small amounts of other metabolites. Thus, plasma exposure to drug-derived radioactivity was primarily due to parent JTZ-951, and the drug was cleared mainly by excretion of unchanged JTZ-951. The study appropriately characterized the disposition of JTZ-951 in patients with end-stage renal disease.

Discovery of Clinical Candidate (5-(3-(4-Chlorophenoxy)prop-1-yn-1-yl)-3-hydroxypicolinoyl)glycine, an Orally Bioavailable Prolyl Hydroxylase Inhibitor for the Treatment of Anemia.

The design and discovery of a new series of (5-alkynyl-3-hydroxypicolinoyl)glycine inhibitors of prolyl hydroxylase (PHD) are described. These compounds showed potent in vitro inhibitory activity toward PHD2 in a fluorescence polarization-based assay. Remarkably, oral administration of 17, with an IC50 of 64.2 nM toward PHD2, was found to stabilize HIF-α, elevate erythropoietin (EPO), and alleviate anemia in a cisplatin-induced anemia mouse model with an oral dose of 25 mg/kg. Rat and dog studies showed that 17 has good pharmacokinetic properties, with oral bioavailabilities of 55.7 and 54.0%, respectively, and shows excellent safety profiles even at a high dose of 200 mg/kg in these animals. Based on these results, 17 is currently being evaluated in a phase I clinical trial for anemia.

A Single-Dose, Open-Label, Randomized, Two-Way Crossover Study in Healthy Japanese Participants to Evaluate the Bioequivalence and the Food Effect on the Pharmacokinetics of Daprodustat.

Daprodustat is a prolyl hydroxylase inhibitor that stimulates erythropoiesis in a manner similar to the natural response to hypoxia, whereby inhibition of hypoxia inducible factor (HIF) prolyl-4-hydroxylases by daprodustat ultimately results in increased levels of HIF-responsive genes. Daprodustat is under development as an emerging new class of agents for the treatment of anemia associated with chronic kidney disease (CKD). This was a single-center, single-dose, open-label, randomized, 2-way crossover study in healthy Japanese male participants consisting of 2 parts. The primary objective was to evaluate the bioequivalence (BE) between daprodustat tablet strengths (part 1) and to evaluate the food effect on the pharmacokinetics (PK) of daprodustat (part 2). A total of 64 healthy Japanese male participants were enrolled; 52 participants were included in part 1 and 12 in part 2. BE was demonstrated between the daprodustat 2-mg tablet and the daprodustat 4-mg tablet. A standard CKD meal did not have a large effect on the PK parameters of daprodustat after a single oral dose of daprodustat 4 mg. Administration of single oral doses of daprodustat 4 mg was generally well tolerated in the healthy Japanese participants, and no new safety signals were identified without regard to food.

Oral Hypoxia-Inducible Factor Prolyl Hydroxylase Inhibitor Roxadustat (FG-4592) for Treatment of Anemia in Chronic Kidney Disease: A Placebo-Controlled Study of Pharmacokinetic and Pharmacodynamic Profiles in Hemodialysis Patients.

Roxadustat (FG-4592), an oral hypoxia-inducible factor prolyl hydroxylase inhibitor that stimulates erythropoiesis, was evaluated in a phase 1b study in patients with end-stage renal disease with anemia on hemodialysis. Seventeen patients, on epoetin-alfa maintenance therapy with stable hemoglobin levels ≥10 g/dL, had epoetin-alfa discontinued on day 3 and were enrolled in this double-blind placebo-controlled study. Two cohorts were randomized 3:1 (roxadustat: placebo). Patients received single doses of roxadustat (1 or 2 mg/kg) or placebo 1 hour after hemodialysis on day 1 and 2 hours before dialysis on day 8. Maximum plasma concentration and area under the plasma concentration-time curve for patients receiving roxadustat were slightly more than dose proportional and elimination half-life ranged from 14.7 to 19.4 hours. Roxadustat was highly protein bound (99%) in plasma, and dialysis contributed a small fraction of the total clearance: only 4.56% and 3.04% of roxadustat recovered from the 1 and 2 mg/kg dose groups, respectively. Roxadustat induced transient elevations of endogenous erythropoietin that peaked between 7 and 14 hours after dosing and returned to baseline by 48 hours after dosing. Peak median endogenous erythropoietin levels were 96 mIU/mL and 268 mIU/mL for the 1- and 2-mg/kg doses, respectively, within physiologic range of endogenous erythropoietin responses to hypoxia at high altitude or after blood loss. No serious adverse events were reported, and there were no treatment- or dose-related trends in adverse event incidence.

Roxadustat: First Global Approval.

Roxadustat (Ai Rui Zhuo® in China) is an orally administered, small molecule hypoxia-inducible factor (HIF) prolyl hydroxylase inhibitor that is being developed by FibroGen, in collaboration with Astellas and AstraZeneca, for the treatment of anaemia in patients with dialysis-dependent chronic kidney disease (CKD), non-dialysis-dependent CKD and in patients with myelodysplastic syndromes. The drug reversibly binds to and inhibits HIF-prolyl hydroxylase enzymes that are responsible for the degradation of transcription factors in the HIF family under normal oxygen conditions. Inhibition of these enzymes reduces HIF breakdown and promotes HIF activity, leading to an increase in endogenous erythropoietin production, thereby enhancing erythropoiesis. It also reduces the expression of the peptide hormone hepcidin, improves iron availability and increases haemoglobin levels. HIF regulates the expression of genes in response to reduced oxygen levels, including genes required for erythropoiesis and iron metabolism. Roxadustat is approved in China and is under regulatory review in Japan for the treatment of anaemia in patients with dialysis-dependent CKD. Studies are underway to investigate long-term cardiovascular outcomes with roxadustat versus placebo (for non-dialysis-dependent CKD) or standard of care (for dialysis-dependent CKD). This article summarizes the milestones in the development of roxadustat leading to this first approval.

Phase I Clinical Study of ZYAN1, A Novel Prolyl-Hydroxylase (PHD) Inhibitor to Evaluate the Safety, Tolerability, and Pharmacokinetics Following Oral Administration in Healthy Volunteers.

OBJECTIVE: This phase I study of ZYAN1 was conducted to evaluate the safety, tolerability, and pharmacokinetics following oral administration in healthy volunteers. METHODS: The study was a randomized, double-blind, placebo-controlled phase I study carried out in two parts in addition to a third part involving an open-label study to evaluate the food/sex effect. A total of 100 subjects were enrolled into the study as follows: part I-single-dose study with ZYAN1 10, 25, 50, 100, 150, 200, and 300 mg (n = 56); part II-multiple-dose study with every other day dosing of ZYAN1 100, 150, 200, and 300 mg (n = 32); and part III-sex and food effect study with ZYAN1 150 mg (n = 12; open-label). RESULTS: ZYAN1 was well-tolerated after single and multiple oral ascending doses. No drug-related serious adverse events were reported. Following a single ascending dose of ZYAN1, the maximum concentration (C max) ranged from 566.47 ± 163.03 to 17,858.33 ± 2899.19 ng/mL and the median time to C max (t max) was approximately 2.5 h for the studied 30-fold oral doses of ZYAN1. Regardless of single or multiple doses, mean C max and area under the concentration-time curve from time zero to time t (AUC t ) values generally showed a dose-proportional increase. The mean elimination half-life (t ½) of ZYAN1 ranged from 6.9 to 13 h with negligible accumulation. Following a single dose of ZYAN1, the mean serum erythropoietin (EPO) C max values showed dose response (i.e., 6.6 and 79.9 mIU/L for 10 and 300 mg ZYAN1 doses, respectively), while the time to mean maximal serum EPO concentrations ranged from 10 to 72 h. CONCLUSION: Oral single (10-300 mg) and multiple dosing (100-300 mg) of ZYAN1 in healthy subjects was found to be safe and well-tolerated. With increasing ZYAN1 dose, there was almost a proportional increase in mean C max and AUC t . The mean serum EPO concentrations showed a trend of dose response. Based on the t ½, pharmacodynamic activity, and lack of drug accumulation, a once every 2 days dosing regimen of ZYAN1 was appropriate for phase II study. TRIAL REGISTRATION: Australian New Zealand Clinical Trials Registry trial ID ACTRN12614001240639.

Effect of Moderate Hepatic Impairment on the Pharmacokinetics and Pharmacodynamics of Roxadustat, an Oral Hypoxia-Inducible Factor Prolyl Hydroxylase Inhibitor.

BACKGROUND AND OBJECTIVE: Roxadustat is a hypoxia-inducible factor prolyl hydroxylase inhibitor in phase III development for the treatment of anaemia associated with chronic kidney disease. This study evaluated the effects of moderate hepatic impairment on roxadustat pharmacokinetics, pharmacodynamics and tolerability. METHODS: This was an open-label study in which eight subjects with moderate hepatic impairment (liver cirrhosis Child-Pugh score 7-9) and eight subjects with normal hepatic function (matched for body mass index, age and sex) received a single oral 100 mg roxadustat dose under fasted conditions. Blood samples were collected until 144 h post-dose in subjects with moderate hepatic impairment and until 96 h post-dose in subjects with normal hepatic function. RESULTS: In subjects with moderate hepatic impairment, area under the concentration-time curve (AUC) from the time of drug administration to infinity (AUC∞) and observed maximum concentration (C max) were 23 % higher [geometric least-squares mean ratio (GMR) 123 %; 90 % CI 86.1-175] and 16 % lower (GMR 83.6 %; 90 % CI 67.5-104), respectively, than in subjects with normal hepatic function. Mean terminal half-life (t ½) appeared to be longer (17.7 vs. 12.8 h) in subjects with moderate hepatic impairment, however intersubject variability on apparent total systemic clearance after single oral dosing (CL/F), apparent volume of distribution at equilibrium after oral administration (V z/F) and t ½ was approximately twofold higher. Erythropoietin (EPO) baseline-corrected AUC from administration to the last measurable EPO concentration (AUCE,last) and maximum effect (E max) were 31 % (GMR 68.95 %; 90 % CI 29.29-162.29) and 48 % (GMR 52.29 %; 90 % CI 28.95-94.46) lower, respectively, than in subjects with normal hepatic function. The single oral roxadustat dose was generally well tolerated. CONCLUSIONS: This study demonstrated the effect of moderate hepatic impairment on the pharmacokinetics and pharmacodynamics of roxadustat relative to subjects with normal hepatic function. These differences are not expected to be of clinical significance.

The Isoquinolone Derived Prolyl Hydroxylase Inhibitor ICA Is a Potent Substrate of the Organic Anion Transporters 1 and 3.

OBJECTIVE: Many cellular responses to hypoxia are mediated by the transcription factor complex hypoxia-inducible factor (HIF). HIF stability is governed by a family of dioxygenases called HIF prolyl hydroxylases (PHDs). Isoquinolone-derived PHD inhibitors, like 2-(1-chloro-4-hydroxyisoquinoline-3-carboxamido) acetate (ICA), which stabilize the intracellular HIF-α have been suggested as a potentially beneficial therapeutic strategy for the treatment of disorders associated with ischemia. To stabilize HIF-α, ICA has to be taken up into proximal tubule cells (PCTs) across the basolateral membrane by one of the organic anion transporters 1, 2 or 3 (OAT1, OAT2 or OAT3). The release into the urine across the luminal membrane may be mediated by OAT4. METHOD: To demonstrate interaction of ICA with human OAT1, OAT2, OAT3 and OAT4, ICA was tested on these transporters stably transfected in HEK293 cells by using p-aminohippurate (PAH), cGMP and estrone-3-sulfate (ES) as reference substrates, respectively. RESULTS: Uptakes of PAH and ES in OAT1- and OAT3-transfected HEK293 cells were inhibited by ICA with half-maximal inhibition values of 0.29 ± 0.05 and 2.58 ± 0.16 µM, respectively. OAT2 was less sensitive to ICA. Efflux experiments identified ICA as an OAT1 and OAT3 substrate. Preloading OAT4-transfected HEK293 cells with ICA stimulated ES uptake by 18.3 ± 3.8%. CONCLUSION: The uptake of ICA across the basolateral membrane of PCTs occurs mainly by OAT1 and the efflux into the tubular lumen by OAT4.

Release kinetics of prolyl hydroxylase inhibitors from collagen barrier membranes.

Collagen barrier membranes are used in guided tissue regeneration to support healing. This strategy, however, relies on the healing capacity of the tissue. Pharmacological inhibitors of prolyl hydroxylases can support regeneration by enhancing angiogenesis and are therefore a promising tool for periodontology. Here we evaluate the release kinetics of the prolyl hydroxylase inhibitors dimethyloxalylglycine and L-mimosine from collagen barrier membranes. Dimethyloxalylglycine and L-mimosine were lyophilized onto the collagen barrier membranes. The morphology of the collagen barrier membranes was analysed using scanning electron microscopy. The release of prolyl hydroxylase inhibitors was assessed by colorimetric and spectroscopic methods. Their ability to induce a cellular response was assessed in bioassays with gingival and periodontal ligament fibroblasts based on vascular endothelial growth factor production, proliferation, and metabolic activity of the cells. We found that loading of collagen barrier membranes with prolyl hydroxylase inhibitors did not change the overall membrane morphology. Assessment of the release kinetics by direct measurements and based on vascular endothelial growth factor production showed that supernatants obtained from the collagen barrier membranes in the first 6 hours had a sufficient level of prolyl hydroxylase inhibitors to induce vascular endothelial growth factor production. A similar kinetic was found when cell proliferation was assessed. Changes in metabolic activity did not reach the level of significance in the MTT assay. In conclusion, collagen barrier membranes can release prolyl hydroxylase inhibitors thereby increasing the pro-angiogenic capacity of periodontal cells in vitro. These findings provide the basis for preclinical studies to evaluate the regenerative capacity of prolyl hydroxylase inhibitors in periodontology and oral surgery.

Prolyl hydroxylase inhibition corrects functional iron deficiency and inflammation-induced anaemia in rats.

BACKGROUND AND PURPOSE: Small-molecule inhibitors of prolyl hydroxylase (PHD) enzymes are a novel target for the treatment of anaemia and functional iron deficiency (FID). Other than being orally bioavailable, the differentiation of PHD inhibitors from recombinant human erythropoietin (rhEPO) has not been demonstrated. EXPERIMENTAL APPROACH: JNJ-42905343 was identified and characterized as a novel inhibitor of PHD and its action was compared with rhEPO in healthy rats and in a rat model of inflammation-induced anaemia and FID [peptidoglycan-polysaccharide (PGPS) model]. KEY RESULTS: Oral administration of JNJ-42905343 to healthy rats increased the gene expression of cytochrome b (DcytB) and divalent metal-ion transporter 1 (DMT1) in the duodenum, and increased plasma EPO. Repeated administration of JNJ-42905343 for 28 days increased blood haemoglobin, mean corpuscular haemoglobin (MCH) and mean corpuscular volume (MCV). The serum iron concentration was increased with low doses (0.3 mg·kg(-1) ) but reduced at high doses (6 mg·kg(-1) ). In PGPS-treated rats, administration of JNJ-42905343 for 28 days corrected FID and anaemia, as reflected by increased blood haemoglobin, MCH and MCV. Increased expression of DcytB and DMT1 genes in the duodenum resulting in increased iron availability was defined as the mechanism for these effects. rhEPO did not affect DcytB and DMT1 and was not effective in PGPS-treated rats. CONCLUSIONS AND IMPLICATIONS: PHD inhibition has a beneficial effect on iron metabolism in addition to stimulating the release of EPO. Small-molecule inhibitors of PHD such as JNJ-42905343 represent a mechanism distinct from rhEPO to treat anaemia and FID.