First evidence of the incorporation of daprodustat and other hypoxia-inducible factor stabilizers into equine hair by passive transfer based on segmental quantitative analysis.

Daprodustat is a hypoxia-inducible factor prolyl hydroxylase domain (HIF-PHD) inhibitor and is used as an erythropoiesis stimulant for the treatment of anemia in humans. In general, administering daprodustat to horses will result in a lifetime ban from both equestrian sports and horseracing by the International Federation of Horseracing Authorities and the Fédération Équestre Internationale, respectively. To control the misuse/abuse of daprodustat, we conducted nasoesophageal administration of daprodustat (100 mg/day for 3 days) to three thoroughbred mares and the post-administration hair samples collected from the three horses over 6 months were analyzed to demonstrate the potential longer-term detection of daprodustat and its metabolites in hair compared with the detection times of daprodustat of 1 and 2 weeks in plasma and urine respectively. The results of the quantitative 2-cm segmental analysis showed that daprodustat was primarily localized in the proximal region (0-2 cm) at 0.375-0.463 pg/mg at 1 month post-administration. These drug bands were gradually spread out along the hair shaft at a rate consistent with the reported growth rate of horse mane hair (approximately 2.5 cm/month) over the following 6 months. In addition, to attain deeper insight into the mechanism of drug incorporation into hair, a total of 11 relevant parameters, including the actual PK parameters and simulated physicochemical and biopharmaceutical parameters for three HIF stabilizers (i.e., daprodustat, vadadustat, and IOX4), were investigated after normalization of the z-scores of all these parameters. Multiple regression analysis indicated that the major factors contributing to the incorporation of the three drugs into hair were their maximum plasma concentrations and lipophilicities, strongly suggesting that the three HIF stabilizers permeated from the bloodstream into the hair bulb via passive transfer with concentration gradients. This work is the first reported evidence showing the incorporation of HIF stabilizers into hair via passive transfer. In addition, cross-species comparison of drug incorporations into hair between daprodustat in horse and roxadustat in human was made in order to have a better understanding of the interactive interpretations about the analysis results obtained from different species. The above findings are not only useful and beneficial for the purpose of doping control but also provide a better understanding of the mechanism of drug incorporation into horse hair.

Development of a colorimetric α-ketoglutarate detection assay for prolyl hydroxylase domain (PHD) proteins.

Since the discovery of the prolyl hydroxylases domain (PHD) proteins and their canonical hypoxia-inducible factor (HIF) substrate two decades ago, a number of in vitro hydroxylation (IVH) assays for PHD activity have been developed to measure the PHD-HIF interaction. However, most of these assays either require complex proteomics mass spectrometry methods that rely on the specific PHD-HIF interaction or require the handling of radioactive material, as seen in the most commonly used assay measuring [14C]O2 release from labeled [14C]α-ketoglutarate. Here, we report an alternative rapid, cost-effective assay in which the consumption of α-ketoglutarate is monitored by its derivatization with 2,4-dinitrophenylhydrazine (2,4-DNPH) followed by treatment with concentrated base. We extensively optimized this 2,4-DNPH α-ketoglutarate assay to maximize the signal-to-noise ratio and demonstrated that it is robust enough to obtain kinetic parameters of the well-characterized PHD2 isoform comparable with those in published literature. We further showed that it is also sensitive enough to detect and measure the IC50 values of pan-PHD inhibitors and several PHD2 inhibitors in clinical trials for chronic kidney disease (CKD)-induced anemia. Given the efficiency of this assay coupled with its multiwell format, the 2,4-DNPH α-KG assay may be adaptable to explore non-HIF substrates of PHDs and potentially to high-throughput assays.

Enarodustat: First Approval.

The orally active hypoxia inducible factor-proly hydroxylase (HIF-PH) inhibitor enarodustat (ENAROY®, Japan Tobacco) is being developed as an alternative to injectable erythropoietin stimulating agents such as epoetin and darbepoetin for the treatment of anaemia associated with chronic kidney disease (CKD). The drug is approved in Japan and clinical development is ongoing in the USA and South Korea. This article summarizes the milestones in the development of enarodustat leading to this first approval for anaemia associated with CKD.

Investigation of the metabolites of the HIF stabilizer FG-4592 (roxadustat) in five different in vitro models and in a human doping control sample using high resolution mass spectrometry.

FG-4592 is a hypoxia-inducible factor (HIF) stabilizer, which can increase the number of red blood cells in the body. It has not been approved by regulatory authorities, but is available for purchase on the Internet. Due to its ability to improve the oxygen transportation mechanism in the body, FG-4592 is of interest for doping control laboratories, but prior to this study, little information about its metabolism was available. In this study, the metabolism of FG-4592 was investigated in a human doping control sample and in five in vitro models: human hepatocytes and liver microsomes, equine liver microsomes and S9 fraction and the fungus Cunninghamella elegans. By using liquid chromatography coupled to a Q-TOF mass spectrometer operated in MSE and MSMS modes, twelve different metabolites were observed for FG-4592. One monohydroxylated metabolite was detected in both the human and equine liver microsome incubations. For the fungus Cunninghamella elegans eleven different metabolites were observed of which the identical monohydroxylated metabolite had the highest response. This rich metabolic profile and the higher levels of metabolites produced by Cunninghamella elegans demonstrates its usefulness as a metabolite producing medium. In the doping control urine sample, one metabolite, which was the result of a direct glucuronidation, was observed. No metabolites were detected in neither the human hepatocyte nor in the equine liver S9 fraction incubates.

Effects of intermittent hypoxia and hyperoxia on angiogenesis and lung development in newborn mice.

BACKGROUND: Premature birth disrupts hypoxia driven microvascular development that directs alveolar and lung growth. Changes in oxygen exposure after birth can perturb the regulation of angiogenesis leading to bronchopulmonary dysplasia (BPD). We studied the effects of intermittent hypoxia or hyperoxia on HIF and angiogenic gene expression and lung development in newborn mice. METHODS: Newborn litters were randomized within 12 h of birth to 12% O2 (4 h), 50% O2 (4 h) or 12% O2 (2 h)/50% O2 (2 h) followed by room air (RA) recovery for 20 h. Mice in RA were the control group. The mice were exposed to 6 such cycles (D1-D6) and sacrifice on D7. Whole lung mRNA was isolated and gene expression performed by qRT-PCR (HIF1α/2α/1ß; PHD2, Ang1, Tie2, Vegf, VegfR1 & VegfR2) and analyzed by PCR array data analysis web portal. HIF-1α, prolyl hydroxylase-2 and VEGF protein were analyzed in whole lung by ELISA. Lung morphology was assessed by H&E sections and radial alveolar counts; cell proliferation by Ki67 immunostaining. RESULTS: HIF-1α mRNA and VEGF protein were significantly downregulated in the 50% O2 group; VEGF mRNA and protein were significantly downregulated in the 12% O2-50% O2 group; Ang-1 and its receptor mRNA expression were downregulated in 12% O2 and 12% O2-50% O2 groups. 50% O2 (hyperoxia) and 12% O2-50% O2 (hypoxia-hyperoxia) groups demonstrated alveolar simplification by RAC and the same groups had decreased cell proliferation by Ki67 staining compared to RA and hypoxia (12% O2) groups. CONCLUSIONS: Downregulation of HIF and angiogenic gene expression with associated changes in lung histology following intermittent hypoxia-hyperoxia is likely an important contributing factor in the development of BPD.