Chronic preclinical safety evaluation of HematideTM, a pegylated peptidic erythropoiesis stimulating agent in monkeys

Hematide is a synthetic peptide-based, pegylated erythropoiesisstimulating agent in clinical development for treatment of anemia.To support chronic clinical dosing requirements, a 9-month repeatdose IV monkey safety study was undertaken. Animals received0, 0.2, 2 or 20 mg/kg hematide IV every three weeks for ninemonths followed by a 14-week recovery. Hematide administrationwas associated with time and dose-dependent polycythemia. Histologicalfindings were related to exaggerated pharmacology that was secondaryto the administration of an erythropoiesis stimulating agentto a normocythemic animal. In conclusion, these results supportthe use of repeated administration of hematide for the correctionof anemia.

Key words: erythropoiesis, hematopoiesis, pharmacology, safety, pharmacokinetics.

Introduction

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Introduction

Anemia is prevalent in patients with chronic renal failure (CRF),¹and is associated with increased morbidity.² Hematide is a syntheticpeptide-based, PEGylated ESA that was designed to specificallybind and activate the erythropoietin (EPO) receptor resultingin production of red blood cells.^3,4 Hematide’s aminoacid sequence is unrelated to EPO and, as a result is not likelyto induce a cross-reactive immune response against either recombinantor endogenous EPO. In the clinical setting, recombinant EPOESAs have been associated with the development of anti-EPO antibody-mediatedpure red cell aplasia (PRCA).⁵ By virtue of this lack of immunologicalcross-reactivity, hematide corrects anemia in rats with PRCA,⁵and restores Hgb to the target range in PRCA patients whileeliminating the need for red blood cell transfusions.⁶

This is the first study in which an exogenous ESA has been evaluatedfollowing long-term administration to normo-cythemic adult animalswithout data being confounded by extramedullary hematopoiesis,as occurs in rodents,^4,7,8 and immunogenic interference, asis the case for human EPO variants.^8,9 Although transgenic miceexpressing the human EPO gene are able to tolerate chronic polycythemia,due to developmental conditioning (increased plasma nitric oxidelevels and blood viscosity regulation),^7,10 they are not subjectto pulsatile reticulocyte and associated changes caused by repeatinjections administered to animals that underwent normal development.The anemia in chronic renal failure (CRF) patients requireslong term therapeutic support. Therefore a comprehensive non-clinicalsafety program was undertaken including a 9-month IV safetystudy in monkeys.

Design and Methods

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Design and Methods

All animals received care in compliance with the Guide for theCare and Use of Laboratory Animals (NIH Publication, 1996) andthe study was conducted under the auspices of an Internal AnimalCare and Use Committee. Ninety-six experimentally naïveCynomolgus monkeys (Covance, Denver, PA, USA; 2 years old),received 0, 0.2, 2, and 20 mg/kg IV every three weeks (Q3W)for up to nine months followed by a 14-week recovery. Four animals/sexin the 0 and 20 mg/kg/dose groups were sacrificed on Day 90.Four animals/sex in all dose groups were sacrificed on Day 195and on Day 279. The remaining four animals/sex in the 0 and20 mg/kg/dose groups (only three females in Group 4 due to anearly death) were euthanized on Day 372. Clinical observationsincluding ophthalmological examinations, body weight, and foodconsumption were recorded. Clinical pathological evaluations(hematology, blood chemistry, and urinalysis), organ weight,histopathological, and electrocardiographic examinations wereperformed. Determination of plasma drug levels and detectionof anti-hematide antibodies were performed by enzyme-linkedimmunosorbent assays (ELISAs) as previously described.^5,11

Results and Discussion

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Results and Discussion

Clinical observations
There were generally no test article-related effects on bodyweight, mean food consumption, or electrocardiographic measurementsincluding QTc interval.

Erythropoietic pharmacology
Hematologic changes occurred that were consistent with the knownerythropoietic pharmacological action of an ESA (Figure 1).Changes in reticulocytes were synchronous with dosing for the0.2 and 2 mg/kg groups and were elevated throughout the dosingperiod for the 20 mg/kg group. Hematide significantly increasedhemoglobin (Hgb) production. At Day 279, Hgb levels for 0.2.2.0, and 20 mg/kg males were 2.2, 8.4, and 9.5 g/dL over concurrentcontrols respectively. Following cessation of dosing, Hgb valuessteadily decreased towards control values.

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Figure 1. Dose and time-dependent increases in mean hematologic parameters (± SE) following IV Hematide Q3W dosing in male monkeys at 0 ( ${circ}$ ), 0.2 ( $bullet$ ), 2 ( ${square}$ ) and 20 ( ${blacksquare}$ ) mg/kg. Arrow denotes administration day. For the 0 and 20 mg/kg groups; n=16 (Day 1 through Day 90), n=12 (Day 104 through 195), n=8 (Day 209 through 279), n=4 (Day 302 through 372. For the 0.2 and 2 mg/kg groups; n=8 (Day 1 through Day 195) and n=4 (Day 209 to 279). Similar results were observed in the female monkeys.

Changes in platelets showed a synchronicity with dosing (Figure 1)for the 0.2 and 2 mg/kg animals. High dose animals showed plateletincreases through the first three doses followed by decreasedlevels. Immediate increases, followed by decreases, in WBCswere observed. The changes were not considered toxicologicallyrelevant and are consistent with data from other ESAs. Mechanismsthat may account for these findings include non-specific stimulationof hematopoietic progenitor cells, followed by compensatoryproduction, functional iron deficiency and feedback controlof accelerated erythropoiesis in normocythemic animals.^8,12,13

RBC indices through Day 104 are shown in Figure 2. The increasesin MCV (mean corpuscular volume) and MCH (mean corpuscular hemoglobin)followed by subsequent decreases and then decreases in MCHC(mean corpuscular hemoglobin content) are consistent with anticipatedRBC rheological changes following accelerated erythropoiesisand subsequent iron-restricted erythropoiesis. The early increasein MCH followed by significant decreases (high dose group) inMCH correlates with decreased serum iron levels measured onDay 90 for the high dose animals.

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Figure 2. Red blood cell (RBC) and secondary hematologic indices (MCV [mean corpuscular volume], MCHC [mean corpuscular hemoglobin content] and MCH [mean corpuscular hemoglobin]) profiles for female monkeys following IV Hematide at 0 ( ${circ}$ ), 0.2 ( $bullet$ ), 2 ( ${square}$ ) and 20 ( ${blacksquare}$ ) mg/kg every three weeks for 5 administrations. Data are expressed as mean with arrows depicting administration days. For the 0 and 20 mg/kg groups; N=16 (Day 1 through Day 90), N=10–12 (Day 104) and for the 0.2 and 2 mg/kg groups; N=8. Similar results were generally observed in the male monkeys.

Due to the pronounced polycythemia, recovery of serum from bloodsamples collected on Days 90, 195 and 279 was significantlyreduced or absent from several animals in the 2.0 and 20 mg/kgdose groups. Commonly affected serum chemistry parameters includedincreased aspartate aminotransferase, total bilirubin and potassiumand decreased iron. AST, total bilirubin, and potassium canbe increased following increased erythrocyte destruction and/orhemolysis.⁸ No differences from controls in serum chemistryparameters were observed following the fourteen-week recoveryperiod.

Immunogenicity
Upon initial screening, five of the 32 monkeys in the 20 mghematide/kg group tested positive for anti-hematide antibodies.A specificity retest demonstrated that one animal containedhematide-specific antibodies. Samples from two others had insufficientvolume for retesting and so were classified as likely positives.The remaining two animals were classified as likely negativesbased on antibody binding that was not specific for hematide.No animals contained antibodies reactive with EPO at any timepoint tested. The presence of antibodies did not alter pharmacology,pharmacokinetics or toxicology.

Pharmacokinetics
Hematide induced sustained plasma levels following IV administration(Table 1). Increases in exposure (AUC values) were greater thandose proportional. Apparent half-life (t1/2) at steady state(Day 106) ranged from approximately 30 hours at the low doseto approximately 90 hours at the high dose. There were no gender-relateddifferences. There were no changes in pharmacokinetics afterevery third week dosing at 0.2 and 2 mg/kg for nine months.At a 20 mg/kg dose, a 1.5 fold increase in plasma AUC was notedafter 6-month dosing, which may have been related, in part,to the reduced plasma volume.

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Table 1. Pharmacokinetic parameters of hematide following every three week (Q3W) IV administration in monkeys.

Unscheduled deaths
Unscheduled deaths included 3 high-dose monkeys (20 mg/kg) onDays 79, 104 and 207. No adverse clinical signs preceded thesedeaths. Based on increased erythron, (Hgb levels of 22–25g/dL) hypertension may have been present. Histologically, congestion(accumulation of blood in the microvasculature), in the sternalbone marrow, kidney, liver, lungs, spleen and gastrointestinaltract occurred with the early deaths. Congestion in the choroidplexus of brains from two of the monkeys and inflammation andmyocardial vacuolation of the heart with accompanying inflammationof the third monkey likely contributed to their early death.

Histopathology
No drug-induced alterations were observed in the 0.2 mg/kg groups.Sternal bone marrow hypercellularity and microvasculature congestionwere observed in brain, kidneys, liver, lung, spleen, and gastro-intestinaltract in the 20 mg/kg monkeys from Day 90 and from the 2 and20 mg/kg monkeys from Days 195 and 279. Incidences increasedwith dose. In general, 14 weeks following cessation of dosing(Day 372), the changes had reversed or were trending to normalcy.Unlike rodents and patients with polycythemia vera,⁷ extramedullaryhematopoiesis and splenomegaly were not observed in these monkeys.Splenectomy of transgenic mice overexpressing EPO resulted ina 30% reduction in hematocrit.⁷ This further enforces the clinicalvalue of studying chronic polycythemia in monkeys, a hematopoieticmodel that is more reflective of the human condition.

The toxicological findings associated with chronic dosing ofhematide in monkeys are attributable to an exaggerated pharmacologicalresponse and changes were generally reversed following the 14-weekrecovery period. This is the first reported study in which anESA was able to be administered chronically to animals withoutfindings being confounded by neutralizing antibodies and extramedullaryhematopoiesis.^4,7–9 In the clinical setting, hematidewill be administered to anemic CRF patients at lower doses,and less frequently,¹⁴ than used in this study and, therefore,the sequelae to exaggerated pharmacology is unlikely to occur.In conclusion, hematide is a potent erythropoiesis stimulatingagent exhibiting sustained pharmacological activity, minimalimmunogenicity and toxicity.

Footnotes

Authorship and Disclosures

KWW developed the hypothesis for this study, designed the study,interpreted the data and wrote the manuscript; SDW participatedin the design of the study, interpreted the data and draftedthe manuscript; K-LF participated in the design of the study,analyzed and interpreted the data, and participated in draftingthe manuscript; PJS interpreted the data and participated indrafting the manuscript; TF participated in the design and conductof the study and drafted the manuscript; CBS designed the study,interpreted the data and participated in drafting the manuscript;DN designed and conducted the study, interpreted the data anddrafted the manuscript. KWW and PJS are employees of Affymax.Affymax is developing Hematide^TM for the treatment of anemia.Other authors reported no potential conflicts of interest.

Funding: Kathryn W. Woodburn and Peter J. Schatz are employeesof Affymax Inc. Affymax is developing Hematide for the treatmentof anemia.

Received for publication February 12, 2008. Revision received March 20, 2008. Accepted for publication April 4, 2008.

References

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