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.

Roxadustat Does Not Affect Platelet Production, Activation, and Thrombosis Formation.

Objective: Roxadustat is a new medication for the treatment of renal anemia. EPO (erythropoietin)-the current treatment standard-has been reported to enhance platelet activation and production. However, to date, the effect of roxadustat on platelets is unclear. To address this deficiency, herein, we have evaluated the effect of roxadustat on platelet production and function. Approach and Results: We performed several mouse platelet functional assays in the presence/absence of in vitro and in vivo roxadustat treatment. Both healthy and 5/6 nephrectomized mice were utilized. The effect of roxadustat on platelet function of healthy volunteers and chronic kidney disease patients was also evaluated. For platelet production, megakaryocyte maturation and proplatelet formation were assayed in vitro. Peripheral platelet and bone marrow megakaryocyte counts were also determined. We found that roxadustat could not stimulate washed platelets directly, and platelet aggregation, spreading, clot retraction, and P-selectin/JON/A exposure were similar with or without in vitro or in vivo roxadustat treatment among both healthy and 5/6 nephrectomized mice. In vivo mouse thrombosis models were additionally performed, and no differences were detected between the vehicle and roxadustat treatment groups. EPO, which was considered a positive control in the present study, promoted platelet function and production as reported previously. Megakaryocyte maturation and proplatelet formation were also not significantly different between control mice and those treated with roxadustat. After receiving roxadustat for 14 days, no difference in the peripheral platelet count was observed in the mice. Conclusions: Administration of roxadustat has no significant impact on platelet production and function.

Effect of Erythropoietin on Calcification of Vascular Smooth Muscle Cells and Its Molecular Regulatory Mechanism.

To investigate its effect and molecular regulatory mechanism on vascular calcification, EPO was added to vascular smooth muscle cells cultured in vitro and injected intraperitoneally into SD rats. The effect of EPO on VSMC calcification was determined by alizarin red staining and ALP activity. Differentially expressed genes were screened by transcriptome sequencing and the relationship and function were verified. We found EPO promotes VSMC calcification in vitro and blood calcification in vivo in a dose-dependent manner. A total of 88 upregulated genes and 59 downregulated genes were detected in transcriptome sequencing, among which the expression of genes associated with bone formation exhibited a marked increase, namely the GATA6 transcription factor, BMP2, RUNX2, OPN, and OCN. Dual luciferase assay has indicated that the binding of GATA6 to BMP2 promoter facilitates the transcription of BMP2. Taken together, findings indicate that EPO can enhance the calcification of VSMCs by activating the GATA6/BMP2 signal axis.

New nanoparticles for topical ocular delivery of erythropoietin.

Erythropoietin (EPO) is known for its neuroprotective and neuroregenerative properties. EPO topical ocular administration has not been tested yet and its bioavailability could be improved by mucoadhesive hydrogels. Thus, this study aimed to develop and evaluate a chitosan (CS) and hyaluronic acid (HA) nanoparticulate system for topical ocular delivery of EPO. Nanoparticles were prepared by ionotropic gelation using six different HAs (HA1-HA6), and characterized by size, zeta potential (ZP), polydispersity index (Pdi), cytotoxicity and mucoadhesion. Encapsulation efficiency and drug loading capacity were also determined. Ex vivo permeation was tested using fresh porcine corneas, scleras and conjunctivas. The permeated EPO was quantified by ELISA, and its presence in the membranes was confirmed by immunohistochemistry. Nanoparticles (NPs) presented size ≤300 nm, ZP around +30 mV and low Pdi (0.167-0.539) at a 1:1 CS:HA mass ratio. The most suitable HA was HA6 (300 kDa - Eye), which had the best mucoadhesive properties. CS/HA6-EPO nanoformulation permeated more rapidly through porcine conjunctiva, followed by sclera and thirdly by cornea, as assessed by immunohistochemistry. All formulations were noncytotoxic on ARPE-19 and HaCaT cell lines, as evaluated by metabolic and membrane integrity tests. In conclusion, CS/HA6-EPO NPs could be a promising formulation for increasing EPO ocular bioavailability by enhancing its retention time and permeation through the different ocular membranes.

Chronic preclinical safety evaluation of EPO-018B, a pegylated peptidic erythropoiesis-stimulating agent in monkeys and rats.

EPO-018B, a synthetic peptide-based erythropoiesis stimulating agent (ESA), is mainly designed for treatment of anemia caused by chronic renal failure and chemotherapy against cancer. It overcomes the deficiencies of currently approved ESA, including the frequent administration of temperature-sensitive recombinant protein and anti-EPO antibody-mediated pure red cell aplasia (PRCA). This study was designed to evaluate the potential chronic toxicity of EPO-018B. Subcutaneous administration doses were designed as 0, 0.2, 1 and 10mg/kg for six months for 160 rats (20/gender/group) and 0, 0.3, 3 and 20mg/kg for nine months for 32 monkeys (4/gender/group) once every three weeks. The vehicles received the same volume of physiological saline injection. All animals survived to the scheduled necropsies after six weeks (for rats) and fourteen weeks (for monkeys) recovery period, except for the two high-dose female rats and two high-dose male monkeys, which were considered related to the increased RBCs, chronic blood hyperviscosity and chronic cardiac injury. EPO-018B is supposed to be subcutaneously injected once every month and the intended human therapeutic dose is 0.025mg/kg. The study findings at 0.2mg/kg for rats and 0.3mg/kg for monkeys were considered to be the study NOAEL (the no observed adverse effect level), which were more than ten times the intended human therapeutic dose. Higher doses caused adverse effects related to the liver toxicity, cardiotoxicity, appearance of neutralizing antibodies of EPO-018B and the decrease of serum glucose and cholesterol. Most treatment-induced effects were reversible or revealed ongoing recovery upon the discontinuation of treatment. The sequelae occurred in rats and monkeys were considered secondary to exaggerated pharmacology and would less likely occur in the intended patient population. As to the differences between human beings and animals, the safety of EPO-018B need to be further confirmed in the future clinical studies.

Impaired renal endothelial nitric oxide synthase and reticulocyte production as modulators of hypertension induced by rHuEPO in the rat.

Our aim was to study the effect of a broad range of recombinant human erythropoietin (rHuEPO) doses on hematological and biochemical parameters, blood pressure (BP), renal function and damage in the rat, focusing on endothelial nitric oxide synthase (eNOS) and hypoxia-inducible factors (HIFs). Male Wistar rats were divided in 5 groups receiving different doses of rHuEPO (100, 200, 400 and 600IU/kg body weight (BW)/week) and saline solution (control), during 3weeks. Blood and 24h urine were collected to perform hematological and biochemical analysis. BP was measured by the tail-cuff method. Kidney tissue was collected to mRNA and protein expression assays and to characterize renal lesions. A dose-dependent increase in red blood cells count, hematocrit and hemoglobin levels was found with rHuEPO therapy, in rHuEPO200, rHuEPO400 and rHuEPO600 groups. Increased reticulocyte count was found in rHuEPO400 and rHuEPO600 groups. BP raised in all groups receiving rHuEPO. The rHuEPO200 and rHuEPO600 groups presented increased kidney protein levels of HIF2α, a reduction in kidney protein levels of eNOS, and the highest grade of vascular and tubular renal lesions. Our study showed that rHuEPO-induced hypertension is present before significant hematological changes occur and, therefore, might involve direct (renal) and indirect (hematological) effects, which varies according to the dose used. The presence of renal hypoxia reduces eNOS activity. Excessive erythrocytosis increases blood hyperviscosity, which can be modulated by an increase in reticulocytes. Hypertension leads to early renal damage without alterations in traditional markers of renal function, thus underestimating the serious adverse effects and risks.

Safety and biosimilarity of ior(®) EPOCIM compared with Eprex(®) based on toxicologic, pharmacodynamic, and pharmacokinetic studies in the Sprague-Dawley rat.

This study examined the safety, pharmacodynamic (PD), and pharmacokinetic (PK) biosimilarity of the human recombinant erythropoietin (EPO) products ior(®) EPOCIM and Eprex(®) following a 28-day repeated intravenous dose administration in male and female Sprague-Dawley rats with a 14-day recovery period. Safety profiling was based on clinical observations, clinical pathology, and pathology findings for control rats dosed with vehicle and rats dosed either with 30, 300, and 600 I.U./kg of ior(®) EPOCIM or 600 I.U. of Eprex(®) . Adverse findings for both ior(®) EPOCIM and Eprex(®) were similar and were a consequence of thrombotic events (ulcerative skin lesions, swollen hock joints/lameness, stomach ulcers) and decreased body weight gains, all known adverse reactions to this class of drug in rats. With the exception of stomach ulcers, all other adverse findings were fully reversible. Neither drug stimulated the production of antidrug antibodies. As expected, ior(®) EPOCIM and Eprex(®) both increased reticulocyte, red blood cell, hemoglobin, and hematocrit levels in rats. The PK of EPO following dosing with ior(®) EPOCIM was well behaved and consistent with the literature. The results of this study imply that ior(®) EPOCIM and Eprex(®) had safety profiles, PD responses, and toxicokinetic profiles that were biosimilar.

High hematocrit resulting from administration of erythropoiesis-stimulating agents is not fully predictive of mortality or toxicities in preclinical species.

We conducted a retrospective analysis of publicly available preclinical toxicology studies with erythropoiesis-stimulating agents (ESAs) to examine common adverse events in rats, Beagle dogs, and cynomolgus monkeys. Mortality and/or thrombotic events were reported sporadically in a subset of studies and attributed to the high hematocrit (HCT) achieved in the animals. However, similarly high HCT was achieved in both high-dose and low-dose groups, but there were no reported adverse events in the low-dose group suggesting HCT was not the sole contributing factor leading to toxicity. Our analysis indicated that increased dose, dose frequency, and dosing duration in addition to high HCT contributed to mortality and thrombosis. To further evaluate this relationship, the incidence of toxicities was compared in rats administered an experimental hyperglycosylated analog of recombinant human erythropoietin (AMG 114) at varying dosing schedules in 1-month toxicity studies. The incidence of mortality and thrombotic events increased in higher dose groups and when dosed more frequently, despite a similarly high HCT in all animals. The results from the investigative study and retrospective analysis demonstrate that ESA-related toxicities in preclinical species are associated with dose level, dose frequency, and dosing duration, and not solely dependent upon a high HCT.

Dose-related differences in the pharmacodynamic and toxicologic response to a novel hyperglycosylated analog of recombinant human erythropoietin in Sprague-Dawley rats with similarly high hematocrit.

We recently reported results that erythropoiesis-stimulating agent (ESA)-related thrombotic toxicities in preclinical species were not solely dependent on a high hematocrit (HCT) but also associated with increased ESA dose level, dose frequency, and dosing duration. In this article, we conclude that sequelae of an increased magnitude of ESA-stimulated erythropoiesis potentially contributed to thrombosis in the highest ESA dose groups. The results were obtained from two investigative studies we conducted in Sprague-Dawley rats administered a low (no thrombotic toxicities) or high (with thrombotic toxicities) dose level of a hyperglycosylated analog of recombinant human erythropoietin (AMG 114), 3 times weekly for up to 9 days or for 1 month. Despite similarly increased HCT at both dose levels, animals in the high-dose group had an increased magnitude of erythropoiesis measured by spleen weights, splenic erythropoiesis, and circulating reticulocytes. Resulting prothrombotic risk factors identified predominantly or uniquely in the high-dose group were higher numbers of immature reticulocytes and nucleated red blood cells in circulation, severe functional iron deficiency, and increased intravascular destruction of iron-deficient reticulocyte/red blood cells. No thrombotic events were detected in rats dosed up to 9 days suggesting a sustained high HCT is a requisite cofactor for development of ESA-related thrombotic toxicities.

Evaluation of antiproliferative and antimetastatic effects of heparin and erythropoietin on B16f10 melanoma cell line.

A number of chemotherapeutic agents and treatment strategies have been developed or designed to treat cancer patients. However, chemotherapeutic regimens frequently cause side-effects, one of which is anemia, a severe clinical problem for cancer patients. Erythropoietin is commonly used to treat anemia and reduce blood transfusions in cancer patients. Another agent which has potential use in cancer therapy is heparin, a glycosaminoglycan with a negative charge, known to increase the clearance of tumor cells from the blood in mice and also has anti-metastatic effects. In this current study, we investigated the effects of rEpo and heparin either as single agents or in combination on B16F10 melanoma cells. Contrary to our expectations based on the previous in vitro and in vivo studies, we have not found a significant growth-promoting effect of rEpo on B16F10 cells. We have also not observed a significant cytotoxic effect of heparin on B16F10 melanoma cell as assayed by MTT test (p > 0.05). However, heparin did significantly prevent the migration/proliferation of B16F10 cell in the wound assay as compared to the control cells after 24 h of incubation (p < 0.001). In addition, this effect of heparin was not prevented when rEpo was present in the medium in the wound assay (p < 0.01 as compared to the control). These results suggest that heparin may have a therapeutic potential as an anti-metastatic drug for cancer.

Short-term intra-nasal erythropoietin administration with low sialic acid content is without toxicity or erythropoietic effects.

The objective of this investigation was to assess the toxicological potential of nasal formulation of erythropoietin with low sialic acid content (Neuro EPO) after 28 days of intra-nasal dosing in rats besides to evaluate the immunogenicity and erythropoietic effect of the test substance. Healthy Wistar rats of both sexes were used for 28 days subacute toxicity and immunogenicity assays. Doses evaluated were 3450, 4830 and 6900 UI/kg/day. The toxicological endpoints examined included animal body weight, food consumption, hematological and biochemical patterns, antibodies determination, selected tissue weights and histopathological examination. Reversibility of toxic effects was evaluated at high dose 14 days after treatment period. Female B6D2F1 mice were used for evaluated erythropoietic effect of the nasal formulation. Hematological endpoints were examined every week during 28 days of intra-nasal dosing of 6900 UI/kg/day. Variations of hematological patterns were not observed after 28 days of intranasal dosing. A slight increase in glucose level of treated animals within the normal range was observed. This effect was not dose related and was reversible. Antibody formation was not observed in any of the test doses. Histopathological examination of organs and tissues did not reveal treatment induced changes. The administration of Neuro EPO in normocythaemic mice did not produce erythropoietic effect. These results suggest that Neuro EPO could be used as a neuroprotective agent, without significant systemic haematological side effects.

Absence of hematological side effects in acute and subacute nasal dosing of erythropoietin with a low content of sialic acid.

The use of human recombinant erythropoietin (EPO) as a neuroprotective agent is limited due to its hematological side effects. An erythropoietin along with a low content of sialic acid (rhEPOb), similar to that produced in the brain during hypoxia, may be used as a neuroprotective agent without risk of thrombotic events. The objective of this investigation was to assess the toxicological potential of a nasal formulation with rhEPOb in acute, subacute and nasal irritation assays in rats. Healthy Wistar rats (Cenp:Wistar) were used for the assays. In an irritation test, animals received 15 µl of rhEPOb into the right nostril. Rats were sacrificed after 24 h and slides of the nasal mucosa tissues were examined. Control and treated groups showed signs of a minimal irritation consisting of week edema and vascular congestion in all animals. In the acute toxicity test, the dose of 47,143 UI/kg was administered by nasal route. Hematological patterns, body weight, relative organ weight, and organ integrity were not affected by single dosing with rhEPOb. In the subacute toxicity test, Wistar rats of both sexes received 6,600 UI/kg/day for 14 days. The toxicological endpoints examined included animal body weight, food consumption, hematological and biochemical patterns, selected tissue weights, and histopathological examinations. An increase of lymphocytes was observed in males that was considered to reflect an immune response to treatment. Histopathological examination of organs and tissues did not reveal treatment-induced changes. The administration of rhEPOb at daily doses of 6,600 UI/kg during 14 days did not produce hematological side effects. These results suggest that rhEPOb could offer the same neuroprotection as EPO, without hematological side effects.

Statins inhibited erythropoietin-induced proliferation of rat vascular smooth muscle cells.

Erythropoietin (EPO) directly stimulates the proliferation of vascular smooth muscle cells, and this is believed to be one of the mechanisms of vascular access failure of hemodialysis patients. However, precise mechanisms of the EPO-induced proliferation of vascular smooth muscle cells are not certain. HMG-CoA reductase inhibitors (statins) are primarily used to reduce cholesterol levels, but also exert other effects, including reno-protective effects. We evaluated the effect of several statins with various hydrophilicities on the EPO-induced proliferation of primary cultured rat vascular smooth muscle cells (VSMCs) in vitro. EPO significantly and concentration-dependently increased DNA synthesis as assessed by [³H]thymidine incorporation, cell proliferation as assessed by WST-1 assay, and activation of the p44/42MAPK pathway. Therapeutic doses of statins (pravastatin, simvastatin, atorvastatin and fluvastatin) in patients with hypercholesterolemia almost completely suppressed all of the EPO-induced effects in a concentration-dependent manner. Co-addition of mevalonic acid almost completely reversed the effects of statins. Statin alone did not affect the basal proliferation capacity of the cells. The effects were almost similar among the statins. We concluded that statins inhibited EPO-induced proliferation in rat VSMCs at least partly through their inhibition of HMG-CoA reductase activity. In the future, statins might prove useful for the treatment of EPO-induced hyperplasia of vascular access. Because the statins all showed comparable effects irrespective of their hydrophilicities, these effects might be a class effect.

Genotoxicity studies on HM10760A, recombinant human erythropoietin conjugated to globin fragment.

HM10760A is a recombinant human erythropoietin chemically conjugated to the N-terminus of human immunoglobulin Fc fragment through a polyethylene glycol linker. HM10760A was shown to have a relatively long half-life, compared with unconjugated recombinant erythropoietin. In this study, the genotoxicity of HM10760A was investigated by using a test battery of three different methods. In the Ames assay, five strains (TA100, TA1535, TA98, TA1537, and Escherichia coli WP2 uvrA) were tested at six concentrations of 3.13, 6.25, 12.5, 25, 50, and 100microg/plate. HM10760A did not increase the number of revertant colonies in any tester strains with and without metabolic activation by rat-liver S9 mix. Subsequently, in vitro chromosomal aberration test, using Chinese hamster lung cells, were conducted at the concentrations of 25, 50, and 100microg/mL. HM10760A did not induce chromosomal aberrations either in the short-period (6 hours) test with or without rat-liver S9 mix or in the continuous-treatment (24 hours) test. In the in vivo bone marrow micronucleus assay using the male ICR (imprinting control region) mouse, HM10760A was subcutaneously administered twice at 24-hour intervals at doses of 0, 150, 300, and 600microg/kg. HM10760A produced a slight, but statistically significant, increase in the frequency of micronucleated polychromatic erythrocytes at 600microg/kg. However, no biological significance was assumed, because this value was within the historical control range. From these findings obtained from the genotoxicity assays performed in this study, it appears unlikely that HM10760A acts as a genotoxic agent in vitro and in vivo.

Nephrogenic systemic fibrosis in rats treated with erythropoietin and intravenous iron.

PURPOSE: To use a rat model for nephrogenic systemic fibrosis (NSF) that was administered high-dose gadodiamide to determine whether the co-administration of erythropoietin (Epo) and intravenous iron potentiated development of skin lesions that are thought to be a marker for the development of NSF. MATERIALS AND METHODS: The local committee for animal research approved this study. High-dose gadodiamide was administered, 2.5 mmol per kilogram of body weight for 20 days, or 500 times the U.S. Food and Drug Administration-approved dose, to four groups of Hannover-Wistar rats: group A, gadodiamide only; B, gadodiamide and Epo; C, gadodiamide and intravenous iron; and D, gadodiamide, Epo, and intravenous iron. The animals were sacrificed 7 days after final injection, and the authors examined dermal histologic findings from each animal and measured metal deposition by using inductively coupled plasma mass spectrometry. To compare the effect of metal deposition and cellularity, a linear mixed effects model was used to fit the data within PROC MIXED modeled with rat-specific random effects, and subsequently a Dunnett adjustment was performed. RESULTS: Rats treated with gadodiamide and both Epo and intravenous iron (group D) had significantly worse skin lesions at gross and histologic analysis (P = .004) compared with the rate treated with gadodiamide only (group A). Group D also had increased levels of deposited gadolinium as measured by means of mass spectrometry (P = .012). CONCLUSION: With a rat model similar to those already existing in the literature, skin changes were more marked in animals exposed to gadodiamide, Epo, and intravenous iron, as opposed to those animals exposed to gadodiamide alone; this experiment suggests that great caution may be warranted when prescribing gadolinium-based contrast agents to patients receiving Epo and intravenous iron.

Erythropoietin promotes deleterious cardiovascular effects and mortality risk in a rat model of chronic sports doping.

Athletes who abuse recombinant human erythropoietin (rhEPO) consider only the benefit to performance and usually ignore the potential short and long-term liabilities. Elevated haematocrit and dehydratation associated with intense exercise may reveal undetected cardiovascular risk, but the mechanisms underlying it remain to be fully explained. This study aimed to evaluate the cardiovascular effects of rhEPO in rats under chronic aerobic exercise. A ten week protocol was performed in four male Wistar rat groups: control--sedentary; rhEPO--50 IU kg(-1), 3 times/wk; exercised (EX)--swimming for 1 h, 3 times/wk; EX + rhEPO. One rat of the EX + rhEPO group suffered a sudden death episode during the week 8. rhEPO in trained rats promoted erythrocyte count increase, hypertension, heart hypertrophy, sympathetic and serotonergic overactivation. The suddenly died rat's tissues presented brain with vascular congestion; left ventricular hypertrophy, together with a "cardiac-liver", suggesting the hypothesis of heart failure as cause of sudden death. In conclusion, rhEPO doping in rats under chronic exercise promotes not only the expected RBC count increment, suggesting hyperviscosity, but also other serious deleterious cardiovascular and thromboembolic modifications, including mortality risk, which might be known and assumed by all sports authorities, including athletes and their physicians.

Pharmacokinetic, tolerability, and bioequivalence comparison of three different intravenous formulations of recombinant human erythropoietin in healthy Korean adult male volunteers: an open-label, randomized-sequence, three-treatment, three-way crossover study.

BACKGROUND: Existing formulations of recombinant human erythropoietin (rhEPO) in Korea contain human serum albumin. To avoid the potential risk of infection by human serum albumin, a new albumin-free rhEPO has been developed. OBJECTIVE: This study was conducted to characterize and compare the pharmacokinetic and safety profiles and the bioequivalence of a newly developed albumin-free rhEPO (Aropotin [TS Corporation, Seoul, South Korea]) with 2 existing rhEPO formulations (Espogen [LG Life Sciences, Seoul, South Korea]; Recormon [Roche, Basel, Switzerland]) with albumin in healthy Korean subjects. METHODS: This was an open-label, randomized-sequence, 3-treatment, 3-way crossover study in which healthy, nonobese (+/-20% of ideal weight), male volunteers between the ages of 19 and 50 years were assigned to 1 of 2 dose levels (50 IU/kg or 100 IU/kg) of 3 formulations. Blood was collected over 32 hours and plasma rhEPO concentrations were determined using a validated enzyme immunoassay. There was a 14-day washout between periods. The pharmacokinetic parameters of the 3 formulations were compared using the bioequivalence criteria of the US Food and Drug Administration, which requires that the 90% CIs of the geometric mean ratios for AUC(0-t), AUC(0-infinity), and C(max) fall within 0.80 to 1.25. Tolerability was evaluated by physical examination with measurements of vital signs, clinical laboratory tests, and electrocardiogram. Subjects were followed up for 2 weeks after the last administration of study drug. RESULTS: Twelve Korean male volunteers were enrolled and completed the study. Six subjects (mean [SD] age, 22.0 [1.7] years; weight, 63.3 [6.2] kg; height, 172.3 [3.5] cm) received a single 50 IU/kg IV bolus dose of study drug and the remaining 6 subjects (mean [SD] age, 23.7 [1.5] years; weight, 66.3 [4.8] kg; height, 174 [4.7] cm) received 100 IU/kg. After a single 50 IU/kg dose, the geometric mean ratio (90% CI) for Aropotin/Espogen was 1.04 (0.91-1.19) IU/L/h for AUC(0-t) and 1.02 (0.89-1.17) IU/L for C(max). The geometric mean ratio (90% CI) for Aropotin/Recormon was 1.01 (0.88-1.15) IU/L/h for AUC(0-t) and 1.01 (0.89-1.16) IU/L for C(max). After a single 100-IU/kg dose, the geometric mean ratio (90% CI) for Aropotin/ Espogen was 0.98 (0.86-1.13) IU/L/h for AUC(0-t) and 0.99 (0.87-1.13) IU/L for C(max). The geometric mean ratio (90% CI) for Aropotin/Recormon was 0.99 (0.861.14) IU/L/h for AUC(0-t) and 0.96 (0.84-1.10) IU/L for C(max). The most frequent adverse events (AEs) were 3 occurrences of elevated serum creatine phosphokinase and serum lactate dehydrogenase levels in the Recormon 100-IU/kg group (n = 3), 3 events of elevated serum lactate dehydrogenase levels in the Espogen 100-IU/kg group (n = 3), and 4 events of elevated serum total bilirubin levels in the Aropotin 100-IU/kg group (n = 3). All formulations were well tolerated with no serious AEs. CONCLUSION: The new formulation of rhEPO met the regulatory criteria for bioequivalence in these healthy Korean adult male volunteers. All formulations were generally well tolerated.

Pharmacokinetic and toxicity study of intravitreal erythropoietin in rabbits.

AIM: To study the pharmacokinetics and toxicity of intravitreal erythropoietin (EPO) for potential clinical use. METHODS: For toxicity study, 4 groups (60 rabbits) with intravitreal injection (IVit) of EPO were studied (10 U, 100 U, or 1,000 U) per eye for single injection and 0.6 U/eye (the designed therapeutic level in rabbits) for monthly injections (6X). Eye examination, flash electroretinogram (ERG), and fluorescein angiography (FA) were carried out before and after injection. The rabbits were killed for histological study at different intervals. For the pharmacokinetic study, after IVit of 5 U EPO into left eyes, 44 rabbits were killed at different intervals, and the EPO levels in vitreous, aqueous, retina and serum were analyzed by enzyme-linked immunosorbent assay. RESULTS: At all of the time points examined, the eyes were within normal limits. No significant ERG or FA change was observed. The histology of retina remained unchanged. The pharmacokinetic profile of EPO in ocular compartments was summarized as follows. The half-life times of EPO in vitreous, aqueous and serum were 2.84, 3.24 and 2.12 d, respectively; and Cmax were 4615.75, 294.31 and 1.60 U/L, respectively. EPO concentrations in the retina of the injected eye peaked at 1.36 U/g protein at 6 h following injection, with the half-life observed to be 3.42 d. CONCLUSIONS: IVit of EPO in a wide range is well tolerated and safe for rabbit eyes. At doses up to 10-fold higher than therapeutic levels, EPO has a pharmacokinetic profile with faster clearance, which is favorable for episodic IVit.

Intravitreal recombinant human erythropoietin: a safety study in rabbits.

PURPOSE: To evaluate the ocular safety of varying doses of a single intravitreal injection of the candidate neuroprotective agent, recombinant human erythropoietin (rhEPO). METHODS: Thirty New Zealand rabbits were divided into one of six groups: untreated controls, intravitreal saline injection, and intravitreal injections of rhEPO (100 U, 250 U, 500 U, or 1000 U). Electroretinography (ERG) was performed one day prior to injection and on post-injection days 3, 7, 14, and 21. Fluorescein angiography was done on post-injection day 28 and graded for the presence of neovascularization by a masked observer. Animals were sacrificed for histologic examination 30 days after injection. RESULTS: Except for the rhEPO 500 U group on day 21, there were no statistically significant differences in the amplitude or implicit time of the ERGs between groups or at different timepoints. Fluorescein angiography showed no evidence of neovascularization. Light microscopy showed no apparent abnormalities in retinal morphology or evidence of retinal damage compared to control groups. CONCLUSION: A single 0.1-ml intravitreal injection of rhEPO at a dose of up to 1000 U does not appear to cause adverse effects on retinal vasculature, retinal anatomy, or ERG function in albino rabbits.