A Full Target-Mediated Drug Disposition (TMDD) Model to Explain the Changes in Recombinant Human Erythropoietin (rhEpo) Pharmacokinetics in Patients with Different Bone Marrow Integrity Following Hematopoietic Transplantation.

Our aim was to build a mechanistic full target-mediated drug disposition (TMDD) model for rhEpo to better understand rhEpo disposition, Epo receptor (EpoR) synthesis, and degradation in hematopoietic transplant patients with four distinct bone marrow conditions. All PK data were analyzed simultaneously using the nonlinear mixed effect modeling approach with NONMEM. The final model was a two-compartmental full TMDD model, which adequately characterizes rhEpo PK in patients and provides insight into the dynamics of free EpoR, rhEpo-EpoR, and total EpoR. The model predicted association rate constant (kon), dissociation rate constant (koff), and internalization rate constant (kint) were 0.0276 pM-1h-1, 0.647 h-1, and 0.255h-1, respectively, which were supported by experimental data. Also, the EpoR degradation rate constant (kdeg) was estimated to be 0.461 h-1. EpoR production rate was estimated to be 37.5 pM/h for adults at pre-ablation baseline and 5.91 pM/h, and 4.19 pM/h in the early post-transplant post-engraftment, and late post-transplant full engraftment. Our model provides extensive information on the dynamics of free EpoR, total EpoR and rhEpo-EpoR, and proven to be more robust and can provide more physiologically relevant binding parameters than previous models.

Chitosan and Hyaluronic Acid Nanoparticles as Vehicles of Epoetin Beta for Subconjunctival Ocular Delivery.

Neuroprotection in glaucoma using epoetin beta (EPOß) has yielded promising results. Our team has developed chitosan-hyaluronic acid nanoparticles (CS/HA) designed to carry EPOß into the ocular globe, improving the drug's mucoadhesion and retention time on the ocular surface to increase its bioavailability. In the present in vivo study, we explored the possibility of delivering EPOß to the eye through subconjunctival administration of chitosan-hyaluronic acid-EPOß (CS/HA-EPOß) nanoparticles. Healthy Wistar Hannover rats (n = 21) were split into 7 groups and underwent complete ophthalmological examinations, including electroretinography and microhematocrit evaluations before and after the subconjunctival administrations. CS/HA-EPOß nanoparticles were administered to the right eye (OD), and the contralateral eye (OS) served as control. At selected timepoints, animals from each group (n = 3) were euthanized, and both eyes were enucleated for histological evaluation (immunofluorescence and HE). No adverse ocular signs, no changes in the microhematocrits (≈45%), and no deviations in the electroretinographies in both photopic and scotopic exams were observed after the administrations (p < 0.05). Intraocular pressure remained in the physiological range during the assays (11-22 mmHg). EPOß was detected in the retina by immunofluorescence 12 h after the subconjunctival administration and remained detectable until day 21. We concluded that CS/HA nanoparticles could efficiently deliver EPOß into the retina, and this alternative was considered biologically safe. This nanoformulation could be a promising tool for treating retinopathies, namely optic nerve degeneration associated with glaucoma.

Detection of recombinant erythropoietin biosimilar Jimaixin™ after administration in healthy subjects.

Jimaixin™ (Jintan Ltd, China) is a biosimilar of recombinant erythropoietin (rEPO) now authorized for therapeutic application in China. With a risk of abuse by athletes, a clear evaluation of its detection using the electrophoretic methods in use in antidoping laboratories was necessary. In a previous work, we showed that Jimaixin™ electrophoretic profile presented slight changes compared with the original drug (first generation rEPO) and that a spike of Jimaixin™ in urine and serum was well identified by SDS-PAGE but with less performance by IEF-PAGE unless a neuraminidase treatment was applied first. The aims of this research were to perform an intravenous administration of Jimaixin™ on three healthy subjects (one microdose [10 IU/kg] and three therapeutic doses [50 IU/kg]) and to evaluate the detection in urine and blood up to 7 days post administration. Analysis of the samples showed that Jimaixin™ detection was complicated by IEF-PAGE due to the loss of the most distinctive basic isoforms. In addition, a neuraminidase treatment did not improve detection (contrary to the observations from spike experiments). On the contrary, Jimaixin™ was very efficiently detected in blood and urine by SDS-PAGE: up to 40 h after a microdose and up to 7 days after the therapeutic doses. The effect of Jimaixin™ on hematological parameters was limited to a clear but transitory increase of the reticulocytes. These data give new elements to better survey a potential misuse of Jimaixin™ by athletes.

Impact of anti-PEG antibody affinity on accelerated blood clearance of pegylated epoetin beta in mice.

Antibodies that bind polyethylene glycol (PEG) can be induced by pegylated biomolecules and also exist in a significant fraction of healthy individuals who have never received pegylated medicines. The binding affinity of antibodies against PEG (anti-PEG antibodies) likely varies depending on if they are induced or naturally occurring. Anti-PEG antibodies can accelerate the clearance of pegylated medicines from the circulation, resulting in loss of drug efficacy, but it is unknown how accelerated blood clearance is affected by anti-PEG antibody affinity. We identified a panel of anti-PEG IgG and IgM antibodies with binding avidities ranging over several orders of magnitude to methoxy polyethylene glycol-epoetin beta (PEG-EPO), which is used to treat patients suffering from anemia. Formation of in vitro immune complexes between PEG-EPO and anti-PEG IgG or IgM antibodies was more obvious as antibody affinity increased. Likewise, high affinity anti-PEG antibodies produced greater accelerated blood clearance of PEG-EPO as compared to low affinity antibodies. The molar ratio of anti-PEG antibody to PEG-EPO that accelerates drug clearance in mice correlates with antibody binding avidity. Our study indicates that the bioactivity of PEG-EPO may be reduced due to rapid clearance in patients with either high concentrations of low affinity or low concentrations of high affinity anti-PEG IgG and IgM antibodies.

A quantitative systems pharmacology model of hyporesponsiveness to erythropoietin in rats.

Recombinant human erythropoietin (rHuEPO) is effective in managing chronic kidney disease and chemotherapy-induced anemia. However, hyporesponsiveness to rHuEPO treatment was reported in about 10% of the patients. A decreased response in rats receiving a single or multiple doses of rHuEPO was also observed. In this study, we aimed to develop a quantitative systems pharmacology (QSP) model to examine hyporesponsiveness to rHuEPO in rats. Pharmacokinetic (PK) and pharmacodynamic (PD) data after a single intravenous dose of rHuEPO (100 IU/kg) was obtained from a previous study (Yan et al. in Pharm Res, 30:1026-1036, 2013) including rHuEPO plasma concentrations, erythroid precursors counts in femur bone marrow and spleen, reticulocytes (RETs), red blood cells (RBCs), and hemoglobin (HGB) in circulation. Parameter values were obtained from literature or calibrated with experimental data. Global sensitivity analysis and model-based simulations were performed to assess parameter sensitivity and hyporesponsiveness. The final QSP model adequately characterizes time courses of rHuEPO PK and nine PD endpoints in both control and treatment groups simultaneously. The model indicates that negative feedback regulation, neocytolysis, and depletion of erythroid precursors are major factors leading to hyporesponsiveness to rHuEPO treatment in rats.

Drug-drug interaction of atazanavir on UGT1A1-mediated glucuronidation of molidustat in human.

Molidustat is an oral inhibitor of hypoxia-inducible factor (HIF) prolyl-hydroxylase enhancing the erythropoietin (EPO) response to HIF; it is in clinical development for the treatment of anaemia related to chronic kidney disease. The predominant role of glucuronidation for molidustat clearance (formation of N-glucuronide metabolite M1) and subsequent renal excretion was confirmed in a human mass balance study, with about 85% of the drug being excreted as M1 in urine. The inhibitory effects of 176 drugs and xenobiotics from various compound classes on the UGT-mediated glucuronidation of molidustat in human liver microsomes (HLMs) were investigated. Based on preclinical findings, glucuronidation of molidustat was predominantly mediated by the 5'-diphospho-glucuronosyltransferase (UGT) isoform UGT1A1. Therefore, atazanavir, which is a potent inhibitor of UGT1A1, was chosen for the evaluation of pharmacokinetics and EPO release following a single oral dose of 25 mg molidustat. Molidustat exposure increased about twofold upon coadministration with atazanavir when considering area under plasma concentration-time curve from zero to infinity (AUC) and maximum plasma concentration (Cmax ). Baseline-corrected increase of EPO was 14% and 34% for Cmax and AUC (calculated over 24 hours), respectively. Coadministration of molidustat and atazanavir was well tolerated.

Development and Application of a Physiologically-Based Pharmacokinetic Model to Predict the Pharmacokinetics of Therapeutic Proteins from Full-term Neonates to Adolescents.

Physiologically-based pharmacokinetic (PBPK) modelling provides an integrated framework to predict the disposition of small molecule drugs in children and is increasingly being used for dose recommendation and optimal design of paediatric studies and in regulatory submissions. Existing paediatric PBPK models can be adopted to describe the disposition of therapeutic proteins (TPs) in children by incorporating information on age-related changes of additional physiological and biological parameters (e.g. endogenous IgG, neonatal Fc receptor, lymph flow). In this study, physiological parameters were collated from literature and evaluated for any age-dependent trends. The age-dependent physiological parameters were used to construct a paediatric PBPK model for TPs. The model was then used to predict the pharmacokinetics of recombinant human erythropoietin (EPO), infliximab, etanercept, basiliximab, anakinra and enfuvirtide in paediatric subjects. The developed paediatric PBPK model predicted the drug concentration-time profiles reasonably well in full-term neonates (clinical PK data only available for EPO), infants, children and adolescents with the ratios of predicted over observed clearance values within 1.5-fold and 25 out of 26 clearance predictions were within 0.8- to 1.25-fold of the observed values. The clinically reported data are required to further assess the predictive accuracy of PK for Fc-containing proteins in term-born children younger than 2 months. This study demonstrates the ability of PBPK models accounting for age-dependent changes in relevant parameters to predict the pharmacokinetics of different types of TPs in paediatrics. The information gained from the PBPK models described here can facilitate our understanding of the complexities of TPs' disposition during growth and development.

The Effect of Size, Maturation, Global Asphyxia, Cerebral Ischemia, and Therapeutic Hypothermia on the Pharmacokinetics of High-Dose Recombinant Erythropoietin in Fetal Sheep.

High-dose human recombinant erythropoietin (rEPO) is a promising potential neuroprotective treatment in preterm and full-term neonates with hypoxic-ischemic encephalopathy (HIE). There are limited data on the pharmacokinetics of high-dose rEPO in neonates. We examined the effects of body weight, gestation age, global asphyxia, cerebral ischemia, hypothermia and exogenous rEPO on the pharmacokinetics of high-dose rEPO in fetal sheep. Near-term fetal sheep on gestation day 129 (0.87 gestation) (full term 147 days) received sham-ischemia (n = 5) or cerebral ischemia for 30 min followed by treatment with vehicle (n = 4), rEPO (n = 8) or combined treatment with rEPO and hypothermia (n = 8). Preterm fetal sheep on gestation day 104 (0.7 gestation) received sham-asphyxia (n = 1) or complete umbilical cord occlusion for 25 min followed by i.v. infusion of vehicle (n = 8) or rEPO (n = 27) treatment. rEPO was given as a loading bolus, followed by a prolonged continuous infusion for 66 to 71.5 h in preterm and near-term fetuses. A further group of preterm fetal sheep received repeated bolus injections of rEPO (n = 8). The plasma concentrations of rEPO were best described by a pharmacokinetic model that included first-order and mixed-order elimination with linear maturation of elimination with gestation age. There were no detectable effects of therapeutic hypothermia, cerebral ischemia, global asphyxia or exogenous treatment on rEPO pharmacokinetics. The increase in rEPO elimination with gestation age suggests that to maintain target exposure levels during prolonged treatment, the dose of rEPO may have to be adjusted to match the increase in size and growth. These results are important for designing and understanding future studies of neuroprotection with high-dose rEPO.

Low doses of recombinant human erythropoietin does not affect C-terminal FGF23 in healthy men.

Recombinant human erythropoietin (rhEpo) can improve human performance, but misuse remains difficult to detect. C-terminal fibroblast growth factor 23 (cFGF23) was recently demonstrated to increase following injection of a single high dose rhEpo, but the effect of more frequent low doses is unknown. Using a randomized double-blind placebo-controlled design, we investigated whether 2 weeks of subcutaneous injections three times a week of 50 IU/kg Eprex (low-dose) or 20 IU/kg Eprex (micro-dose) increase cFGF23 levels compared with saline (placebo) injections in 24 healthy males. Venous blood was sampled at day -3, 0, 1, 3, 11, 14, 18, and 25 of the treatment and analyzed for cFGF23 and erythropoietin concentration ([EPO]). The level of cFGF23 was similar at days -3, 0, 1, 3, 11, 14, 18, and 25 with the low-dose (23 ± 4, 26 ± 5, 23 ± 7, 27 ± 6, 25 ± 8, 24 ± 10, 22 ± 5, and 24 ± 7 RU/mL, respectively), micro-dose (23 ± 6, 25 ± 5, 23 ± 8, 28 ± 9, 27 ± 7, 25 ± 9, 25 ± 5, and 23 ± 6 RU/mL, respectively) and placebo (23 ± 6, 24 ± 6, 26 ± 7, 26 ± 6, 31 ± 6, 31 ± 7, 24 ± 4, and 27 ± 8 RU/mL, respectively) treatment. The correlation coefficient between plasma [EPO] and plasma cFGF23 levels was R2 = 0.01 and insignificant. The results demonstrate that cFGF23 is not sensitive to low doses of subcutaneous rhEpo injections in healthy males.

Dose Correction for a Michaelis-Menten Approximation of a Target-Mediated Drug Disposition Model with a Multiple Intravenous Dosing Regimens.

This study aimed to develop a method for implementing dose correction in a Michaelis-Menten (M-M) approximation of a target-mediated drug disposition (TMDD) model with multiple intravenous (IV) bolus administrations. We derived the formula of a correction factor (Fcorr) for each dose in a multiple IV bolus dosing regimens for M-M model. Fcorr depends on the residual free drug amount prior IV bolus dosing event and dose amount. We conducted a stochastic simulation and estimation (SSE) exercise based on therapeutic antibody PK parameters to evaluate the effect of Fcorr on parameter estimation. Previously published clinical PK data of recombinant human erythropoietin (rHuEPO) from four clinical trials in healthy subjects receiving multiple IV bolus doses were analyzed by both M-M model with and without dose correction (MMC and MMNC) as well as the rapid-binding/quasi-steady-state (RB/QSS) TMDD models. Our results showed that MMNC introduced bias to fixed-effect parameter estimates and overestimated random-effect variables. Compared with MMC, MMNC was not able to adequately characterize the nonlinearity in the PK data of antibody and rHuEPO. The MMC-based simulation demonstrated that thricely weekly 10 IU/kg rHuEPO dosing regimen yielded Fcorr = 0.5. This result suggested that the lower-than-expected exposure for rHuEPO at low dose is due to target binding. An M-M approximation of the TMDD model should include a dose correction to avoid model misfitting and potential bias in the estimated PK parameters.

The role of recombinant Human erythropoietin in neonatal anemia.

AIM: To estimate the blood level of Erythropoietin(EPO) in neonates with anemia of prematurity (APO) and in late hypo-regenerative anemia and to clarify role of EPO in correction of anemia and reducing the number of blood transfusions. METHODS: This study was carried out on 60 neonates divided into; group I (30 preterm neonates) with AOP received EPO (250 IU/kg/dose subcutaneously 3 times weekly for 4 weeks), compared to group II (30 neonates) with AOP treated only with blood transfusion. CBC parameters and transfusion requirements were followed during therapy. Serum level of EPO was measured by ELISA technique. RESULTS: By the end of the 4th week of therapy, there was significant increase in group I post r-Hu EPO compared to group II regarding reticulocyte counts (P < 0.001) leading to rise of the Hb (P < 0.001), Hct levels (P < 0.001) with subsequent reduction in the overall number of blood transfusions (P < 0.001). CONCLUSION: EPO therapy in conjunction with iron, vitamin E and folic acid, stimulated erythropoiesis and significantly reduced the need for blood transfusion in AOP.

Target-mediated disposition population pharmacokinetics model of erythropoietin in premature neonates following multiple intravenous and subcutaneous dosing regimens.

Routine erythropoietin (Epo) therapy for neonatal anemia is presently controversial due to its modest response. We speculate that an important contributor to this modest response is that previous clinical study designs were not driven by rigorous mechanistic and kinetic insights into the complex pharmacokinetics (PK) and pharmacodynamics (PD) of Epo in this population. To address this therapeutic opportunity, we conducted a prospective clinical study to investigate the PK of Epo in very-low-birth-weight (VLBW) premature neonates using a unique Epo dosing algorithm that accounts for complex neonatal erythropoietic physiology. Twenty-seven subjects received up to 10 intravenous or subcutaneous exogenous doses of Epo (600 or 1200 U/kg) during the first 4 weeks of life. Subjects were administered two doses of Epo 1200 U/kg on days 2 and 16, and eight doses of Epo 600 U/kg on days 4, 5, 6, 7, 9, 14, 15, and 28 following birth. We have developed for the first time a mechanistic, target-mediated disposition model that provides novel insights into the mechanisms driving Epo PK in VLBW neonates. Epo association rate, kon, was estimated to be 0.00610 pM-1h-1, and the dissociation rate koff was 0.112 h-1. Internalization of the Epo-target complex (kint) and the total receptor concentration (Rmax) were estimated to be 0.118 h-1 and 133 pM, respectively. Following s.c. administration, the absorption rate (ka) of Epo was 0.0738h-1 and bioavailability was 78.0%. Our mechanism-based population pharmacokinetic analysis provided quantitative insight into Epo kinetics in VLBW neonates; the information gained will assist in deriving dosing strategies for neonatal anemia and for neuroprotection efficacy studies.

Plasma Pharmacokinetics of High-Affinity Transferrin Receptor Antibody-Erythropoietin Fusion Protein is a Function of Effector Attenuation in Mice.

Erythropoietin (EPO) is a potential therapeutic for Alzheimer's disease (AD); however, limited blood-brain barrier (BBB) penetration reduces its applicability as a CNS therapeutic. Antibodies against the BBB transferrin receptor (TfRMAbs) act as molecular Trojan horses for brain drug delivery, and a fusion protein of EPO and TfRMAb, designated TfRMAb-EPO, is protective in a mouse model of AD. TfRMAbs have Fc effector function side effects, and removal of the Fc N-linked glycosylation site by substituting Asn with Gly reduces the Fc effector function. However, the effect of such Fc mutations on the pharmacokinetics (PK) of plasma clearance of TfRMAb-based fusion proteins, such as TfRMAb-EPO, is unknown. To examine this, the plasma PK of TfRMAb-EPO (wild-type), which expresses the mouse IgG1 constant heavy chain region and includes the Asn residue at position 292, was compared to the mutant TfRMAb-N292G-EPO, in which the Asn residue at position 292 is mutated to Gly. Plasma PK was compared following IV, IP, and SQ administration for doses between 0.3 and 3 mg/kg in adult male C57 mice. The results show a profound increase in clearance (6- to 8-fold) of the TfRMAb-N292G-EPO compared with the wild-type TfRMAb-EPO following IV administration. The clearance of both the wild-type and mutant TfRMAb-EPO fusion proteins followed nonlinear PK, and a 10-fold increase in dose resulted in a 7- to 11-fold decrease in plasma clearance. Following IP and SQ administration, the Cmax values of the TfRMAb-N292G-EPO mutant were profoundly (37- to 114-fold) reduced compared with the wild-type TfRMAb-EPO, owing to comparable increases in plasma clearance of the mutant fusion protein. The wild-type TfRMAb fusion protein was associated with reticulocyte suppression, and the N292G mutation mitigated this suppression of reticulocytes. Overall, the beneficial suppression of effector function via the N292G mutation may be offset by the deleterious effect this mutation has on the plasma levels of the TfRMAb-EPO fusion protein, especially following SQ administration, which is the preferred route of administration in humans for chronic neurodegenerative diseases including AD.

Hyperhydration Effect on Pharmacokinetic Parameters and Detection Sensitivity of Recombinant Human Erythropoietin in Urine and Serum Doping Control Analysis of Males.

Excessive fluid intake, that is, hyperhydration, may be adopted by athletes as a masking method during antidoping sample collection to influence the excretion patterns of doping agents and, therefore, manipulate their detection. The aim of this exploratory study was to assess the hyperhydration effect on the detection sensitivity of recombinant human erythropoietin (rHuEPO) by sodium N-lauroyl sarcosinate ("sarkosyl") polyacrylamide gel electrophoresis analysis. The influence of hyperhydration on the serum and urinary pharmacokinetic (PK) profiles of rHuEPO was also investigated. Seven healthy physically active nonsmoking Caucasian males participated in a 31-day clinical study comprising a baseline (days 0, 1-3, and 8-10) and a drug phase (days 15-17, 22-24, and 29-31). Epoetin beta was administered subcutaneously at a single dose of 3000 IU on days 15, 22, and 29. Hyperhydration was applied in the morning on 3 consecutive days (days 1-3, 8-10, 22-24, and 29-31), that is, 0, 24, and 48 h after first fluid ingestion. Water and a commercial sports drink were used as hyperhydration agents (20 mL/kg body weight). Serum and urinary concentration-time profiles were best described by a one-compartment PK model with zero-order absorption. Delayed absorption was observed after hyperhydration and, therefore, lag time was introduced in the PK model. Results showed no significant difference (p > 0.05) on serum or urinary erythropoietin concentrations under hyperhydration conditions. A trend for decreasing volume of distribution and increasing clearance after hyperhydration was observed, mainly after sports drink consumption. However, no significant differences (p > 0.05) due to hyperhydration for any of the serum PK parameters calculated by noncompartmental PK analysis were observed. Renal excretion of endogenous erythropoietin and rHuEPO, as reflected on the urinary cumulative amount, was increased approximately twice after hyperhydration and this supports the nonsignificant difference on the urinary concentrations. Analysis of serum and urine samples was able to detect rHuEPO up to 72 h after drug administration. The detection window of rHuEPO remained unaffected after water or sports drink ingestion. Hyperhydration had no effect on the detection sensitivity of EPO either in serum or urine samples.

A Mechanism-Based Population Pharmacokinetics Model of Erythropoietin in Premature Infants and Healthy Adults Following Multiple Intravenous Doses.

The objective of the current study was to develop a population pharmacokinetics (PK) model for erythropoietin (Epo) in premature infants and healthy adults to characterize the variation in PK, and to study the differences in Epo PK in these 2 populations. Thirteen very low-birth-weight premature infants (<1500 g at birth), and 10 healthy adults received up to 4 intravenous doses of Epo that ranged from 10 to 500 U/kg. The final model had a target-mediated saturable, nonlinear, elimination pathway that incorporated the mechanism of Epo binding to its receptors along with a parallel linear, central elimination pathway. Epo clearance was found to be significantly higher in preterm infants compared to adults. Epo clearance via the nonlinear pathway was found to be much higher in infants; they had an Epo receptor capacity of 133 pM vs 86.6 pM in adults, which is most likely due to the higher erythroid progenitor cell mass per kilogram of body weight in infants. The parallel linear elimination was found to be more dominant in adults, reaching 91% of the total clearance with a 500-U/kg dose compared to just 6.1% of the total clearance following the same dose in preterm infants. Thus, this mechanism-based population PK model revealed that receptor-based nonlinear elimination is the dominant Epo elimination pathway in premature infants, and parallel linear elimination is dominant in adults.

The effects of enteral artificial amniotic fluid-containing erythropoietin on short term outcomes of preterm infants.

Hosseini M, Azampour H, Raeisi S, Behtari M, Valizadeh H, Saboohi R. The effects of enteral artificial amniotic fluid-containing erythropoietin on short term outcomes of preterm infants. Turk J Pediatr 2019; 61: 392-398. Necrotizing Enterocolitis (NEC) is a common devastating gastrointestinal disease, which usually develops in premature infants. Erythropoietin (EPO) as a hematopoietic hormone produced by the kidney can also be naturally found in amniotic fluid and breast milk. There is some evidence that supports the contribution of EPO in the prevention of inflammation and intestinal tissue repair. This study was aimed to determine if oral administration of artificial amniotic fluid with or without EPO would protect preterm infants against NEC and improve the certain neonatal outcomes. In this study, 150 preterm infants with gestational age 28 weeks or less and birth weight 1250 grams or less were enrolled. The infants were divided randomly into 3 groups: 1) Control group (n=50) with routine feeding protocol without any administration; 2) Amniotic fluid group (n=50) with 5mL/kg synthetic amniotic fluid; 3) EPO group (n=50) with RhuEPO dissolved in the synthetic amniotic fluid. The administrations of the study solution were started 3 days after the birth and were continued for 3 weeks (21 days). The infants in the study groups were followed up until discharge and the frequency of NEC, mortality, and other complications of the disease among the groups were compared. The mortality rate in preterm infants of the amniotic fluid and EPO groups were significantly lower than in the control group (p=0.027). We couldn`t find any significant differences in the frequency of NEC and other complications among the three study groups. The administration of synthetic amniotic fluid (with or without EPO) in preterm infants may decrease the mortality rate. Use of EPO in synthetic amniotic fluid did not affect the frequency of NEC.

Synergetic Induction of NGF With Diazoxide and Erythropoietin Attenuates Spinal Cord Ischemic Injury.

BACKGROUND: Paraplegia remains a significant complication of thoracoabdominal aortic intervention. We previously reported that diazoxide (DZ), enhances the neuroprotective efficacy of erythropoietin (EPO). We hypothesized that DZ and EPO combined treatment attenuates spinal cord ischemic injury through upregulation of nerve growth factor (NGF). METHODS: DZ (pretreatment) was given to adult male C57/BL6 mice by oral gavage and EPO (before surgery) was intraperitoneally injected 32 h after administration of DZ. Spinal cords were harvested 0, 2, 4, and 6 h after injection of EPO. NGF expression was analyzed by western blot. After determining the optimal time, NGF expression was compared between DZ (pretreatment) + EPO (before surgery), DZ + PBS, PBS + EPO, and PBS + PBS (ischemic control). Four groups were studied to compare the motor function after ischemia: DZ + EPO (n = 11), ischemic control (n = 9), DZ + EPO + tropomyosin receptor kinase A receptor inhibitor (n = 9), and sham (without cross-clamp, n = 4). Spinal cord ischemia was induced by a 4-min thoracic aortic cross-clamp. Functional scoring (Basso Mouse Score) was done at 12-h intervals until 48 h, and spinal cords were harvested for evaluation of NGF expression and histological changes. RESULTS: NGF expression was significantly upregulated 4 h after administration of EPO. At 4 h after injection of EPO, NGF expression in the DZ + EPO group was significantly higher than that in the other groups. DZ + EPO significantly preserved motor function compared with all other groups. At 48 h after reperfusion, the level of NGF expression in the DZ + EPO group, was significantly higher than in all other groups. CONCLUSIONS: DZ + EPO attenuates spinal cord ischemic injury through upregulation of NGF. Better understanding of this mechanism may serve to further prevent ischemic complications for aortic intervention.

Digital PCR detection of plasmid DNA administered to the skeletal muscle of a microminipig: a model case study for gene doping detection.

OBJECTIVE: Doping control is an important and indispensable aspect of fair horse racing; genetic doping has been recently included to this. In this study, we aimed to develop a detection method of gene doping. A plasmid cloned with human erythropoietin gene (p.hEPO, 250 µg/head) was intramuscularly injected into a microminipig. Subsequently, p.hEPO was extracted from 1 mL of plasma and detected by droplet digital polymerase chain reaction. RESULTS: The results confirmed that the maximum amount of plasmid was detected at 15 min after administration and the majority of the plasmid was degraded in the bloodstream within 1-2 days after administration. In contrast, low amounts of p.hEPO were detected at 2-3 weeks after administration. These results suggest that the proposed method to detect gene doping can help obtain information for experiments using horses.

Brain Penetrating Bifunctional Erythropoietin-Transferrin Receptor Antibody Fusion Protein for Alzheimer's Disease.

Erythropoietin (EPO), a glycoprotein cytokine essential to hematopoiesis, has neuroprotective effects in rodent models of Alzheimer's disease (AD). However, high therapeutic doses or invasive routes of administration of EPO are required to achieve effective brain concentrations due to low blood-brain barrier (BBB) penetrability, and high EPO doses result in hematopoietic side effects. These obstacles can be overcome by engineering a BBB-penetrable analog of EPO, which is rapidly cleared from the blood, by fusing EPO to a chimeric monoclonal antibody targeting the transferrin receptor (cTfRMAb), which acts as a molecular Trojan horse to ferry the EPO into the brain via the transvascular route. In the current study, we investigated the effects of the BBB-penetrable analog of EPO on AD pathology in a double transgenic mouse model of AD. Five and a half month old male APPswe/PSEN1dE9 (APP/PS1) transgenic mice were treated with saline ( n = 10) or the BBB-penetrable EPO ( n = 10) 3 days/week intraperitoneally for 8 weeks, compared to same-aged C57BL/6J wild-type mice treated with saline ( n = 8) with identical regiment. At 9 weeks following treatment initiation, exploration and spatial memory were assessed with the open-field and Y-maze test, mice were sacrificed, and brains were evaluated for Aß peptide load, synaptic loss, BBB disruption, microglial activation, and microhemorrhages. APP/PS1 mice treated with the BBB-penetrable cTfRMAb-EPO fusion protein had significantly lower cortical and hippocampal Aß peptide number ( p < 0.05) and immune-positive area ( p < 0.05), a decrease in hippocampal synaptic loss ( p < 0.05) and cortical microglial activation ( p < 0.001), and improved spatial memory ( p < 0.05) compared with APP/PS1 saline controls. BBB-penetrating EPO was not associated with microhemorrhage development. The cTfRMAb-EPO fusion protein offers therapeutic benefits by targeting multiple targets of AD pathogenesis and progression (Aß load, synaptic loss, microglial activation) and improving spatial memory in the APP/PS1 mouse model of AD.