J Vet Intern Med 2004;18:8191

Clinical Efcacy and Safety of Recombinant Canine Erythropoietin in Dogs with Anemia of Chronic Renal Failure and Dogs with Recombinant Human Erythropoietin-Induced Red Cell Aplasia
John F. Randolph, Janet Scarlett, Tracy Stokol, and James N. MacLeod
The efcacy and safety of recombinant canine erythropoietin (rcEPO) therapy was evaluated in 19 dogs with anemia of chronic renal failure (group 1) and 6 dogs with chronic renal failure and recombinant human erythropoietin (rhEPO)-induced red cell aplasia (group 2). Hematocrit (Hct) and absolute reticulocyte count (ARC) were monitored weekly for the rst 8 weeks, CBC (including ARC) and serum iron proles were evaluated monthly, and serum biochemical analyses were performed every 2 months for 6 (group 2) to 12 (group 1) months. For group 1 dogs, median Hct and ARC increased signicantly during the 1st week of rcEPO treatment, and median Hct was sustained at 35% after week 5. In contrast, median Hct and ARC for group 2 did not change signicantly with rcEPO treatment, even with doses greater than those used in group 1. Nevertheless, 2 (33%) of the 6 dogs in group 2 developed erythroid hyperplasia, reticulocytosis, and increases in Hct with rcEPO treatment. Although median systolic blood pressure did not change signicantly in either group, 5 dogs developed systolic blood pressures 180 mm Hg during the study. Appetite and energy level improved in most group 1 dogs with increases in Hct. Recombinant cEPO stimulated erythrocyte production in dogs with nonregenerative anemia secondary to chronic renal failure without causing the profound erythroid hypoplasia that can occur in rhEPO-treated dogs. Unfortunately, rcEPO was not as effective in restoring erythrocyte production in dogs that had previously developed rhEPO-induced red cell aplasia. Key words: Erythroid hypoplasia; Erythropoietin-dependent anemia; Nonregenerative anemia; Uremia.

rythropoietin (EPO) is a glycosylated protein that stimulates erythrocyte production and is produced by peritubular interstitial cells of the renal cortex.13 An absolute or relative deciency of renal EPO production in dogs (and other mammals) with chronic renal failure (CRF) is the main cause of the nonregenerative anemia accompanying uremia.4,5 This EPO-dependent anemia contributes substantially to the clinical signs of weakness, lethargy, and inappetence encountered in patients with CRF.68 To treat anemia of CRF in humans, commercially available recombinant human EPO (rhEPO)a,b is widely used to stimulate erythrocyte production.6,7 Similarly, rhEPO administered to dogs with nonregenerative anemia secondary to CRF induces a rapid and substantial erythrocyte response.912 Unfortunately, in many treated dogs the effect is short lived because of the development of antibodies against rhEPO.1012 Apparently, there is sufcient interspecies variation in EPO structure that the canine immune system can recognize rhEPO as a foreign substance and mount an immune response against the recombinant protein.12,13 The resultant antibodies are thought to block bioactivity of rhEPO and may cross-neutralize residual endogenous canine EPO, leading to the development of life-threatening red cell aplasia.12
From the Departments of Clinical Sciences (Randolph), Population Medicine and Diagnostic Sciences (Scarlett), and Biomedical Sciences (Stokol, MacLeod), College of Veterinary Medicine, Cornell University, Ithaca, NY. Recombinant cEPO is not yet commercially available nor has it received regulatory approval for clinical use in privately-owned dogs outside of the current study. Reprint requests: John F. Randolph, DVM, Diplomate ACVIM, Department of Clinical Sciences, College of Veterinary Medicine, Cornell University, Ithaca, NY 14853; e-mail: jfr4@cornell.edu. Submitted June 16, 2003; Revised August 11, 2003; Accepted September 4, 2003. Copyright 2004 by the American College of Veterinary Internal Medicine 0891-6640/04/1801-0011/$3.00/0

We previously isolated the gene that encodes canine EPO and developed a eukaryotic expression system to produce sufcient quantities of recombinant canine EPO (rcEPO) for therapeutic use.14 Recombinant and native cEPO are structurally identical at the amino acid level, so the problem of immunogenicity seen with rhEPO use in dogs would not be expected with the species-specic rcEPO. Recombinant cEPO effectively stimulated erythrocyte production in clinically normal dogs during a 24-week period without causing the red cell aplasia encountered in rhEPO-treated dogs.15 The purpose of the study reported here was 2-fold: (1) to evaluate the efcacy and safety of rcEPO in dogs with the anemia of CRF and (2) to determine whether rcEPO could restore erythrocyte production in dogs that had developed rhEPO-induced red cell aplasia.

Materials and Methods

Dogs
Twenty-ve privately owned dogs with naturally developing CRF were enrolled in the study between March 1997 and December 2001. Criteria for inclusion in the study included a diagnosis of CRF clearly demonstrated by established standards, nonregenerative anemia (hematocrit [Hct] 25%, absolute reticulocyte count [ARC] 60,000 cells/ L) with no evidence of hemorrhage or hemolysis, and systolic blood pressure of 180 mm Hg. At the start of the study, 2 dogs were enrolled with Hcts of 27.2% and 29.0%. Treatment of the CRF before inclusion in the study included diets formulated for renal failure (16 dogs), oral vitamin and/or mineral supplements (14), H2 receptor blockers (13), phosphate binders (13), subcutaneous uid therapy (9), antihypertensive agents (7), marine oil supplements (5), sodium bicarbonate (4), low-dose aspirin (3), and androgenic steroids (3). Four dogs were receiving thyroid hormone supplement for hypothyroidism, which was satisfactorily controlled based on postsupplement blood thyroxine concentrations. Four dogs were being treated with antibiotics. Nineteen of the 25 dogs had never been treated with rhEPO (group 1); the remaining 6 dogs had developed red cell aplasia as a consequence of previous rhEPO therapy (group 2). The diagnosis of rhEPOinduced red cell aplasia was based on initial increase and then subsequent decrease in Hct accompanied by reticulocytopenia despite con-

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Randolph et al employed for sample analyses with the exception of serum iron proles, which were all produced at the Cornell University College of Veterinary Medicine using automated procedures.e As guidelines for data interpretation, reference ranges established using automated procedurese,f at the Cornell University College of Veterinary Medicine are provided.

tinued or increased rhEPO dosage and bone marrow conrmation (available for 4 dogs) of profound erythroid hypoplasia with a myeloid : erythroid ratio (M : E) of 49 : 1. Group 1 dogs (11 females, 8 males; 9 females and 4 males were neutered) ranged in age from 0.6 to 17 (median, 8) years and had body weights between 7.0 and 38.0 (median, 16.3) kg. Group 2 dogs (5 females, 1 male; all dogs were neutered) ranged in age from 1 to 11 (median, 5) years and had body weights between 6 and 45 (median, 30) kg. Breeds represented more than once included Labrador Retriever (4, group 1), Rottweiler (2, group 2), and Gordon Setter (2, group 1; 1, group 2). Eight dogs were treated at the Cornell University Hospital for Animals; the other 17 dogs were treated at participating veterinary medical teaching hospitals or private veterinary practices. An Internetbased computer program was developed to support collection and input of the primary data from participating veterinary hospitals and to facilitate communication between study coordinators and the collaborating veterinarians. The use of rcEPO was approved by the Institutional Animal Care and Use Committee of Cornell University and sanctioned by the FDA Center for Veterinary Medicine as an investigational new animal drug. Informed consent was provided by each participating dog owner.

Bone Marrow Cytologic Examination

Bone marrow aspirates were collected, stained, and analyzed as previously described.15,16 Slides from all bone marrow aspirates were reviewed by the same clinical pathologist (TS). Bone marrow aspiration was performed before administration of rcEPO (8 dogs, group 1; all 6 dogs, group 2) and at weeks 4 (4, group 1; 4, group 2), 8 (1, group 1; 1, group 2), and 22 (1, group 1) of rcEPO treatment.

Statistical Analyses
Because of the large number of possible comparisons that could be made with these data, the analysis was subdivided into 3 parts. The initial analysis focused on the primary a priori questions involving Hct and reticulocyte response to rcEPO treatment and differences in these results between groups 1 and 2. Signicance was set at P .05 despite multiple comparisons so that important effects would not be missed. The second component of the analysis focused on other potential effects of treatment (eg, declines in mean corpuscular volume [MCV]) that have previously been associated with rhEPO or rcEPO treatment in dogs.12,15 Signicance for these comparisons was similarly kept liberal to facilitate identifying effects in this data set. The third component was an exploration of the data to identify any unanticipated effects that might be important to clinicians using rcEPO. The alpha level (P .001) for these comparisons was kept quite rigorous to minimize the identication of effects that arose by chance because of the multiple comparisons. Recognizing that data obtained from different laboratories may not be directly comparable among dogs, median values were assessed to evaluate general trends in response to rcEPO treatment. Comparisons of continuous hematologic and biochemical values between the 2 groups at baseline were made using the Wilcoxon rank sum test. Medians (and ranges) rather than means were estimated and compared because of the relatively small sample sizes and the lack of normality for the majority of variables evaluated. Comparisons of categorical variables (eg, proportion responding) were made using the chi-square test of independence.17 To evaluate trends within groups over time, median hematologic and biochemical values were rst plotted by week in each group. Then, clinically relevant changes from baseline values were evaluated for signicance using the Wilcoxon signed rank test for only those dogs that had values at each time point. No statistical evaluations were performed for time points with values for 3 dogs. Survival and time to response in the 2 groups were estimated using Kaplan-Meier curves. Comparison of the Kaplan-Meier curves for response to rcEPO treatment was made using the log-rank test.17 The hazard ratio (risk of dying in group 2 compared with that in group 1) was estimated using the Cox proportional hazards model, and the signicance of the comparison was based on the likelihood ratio test.

Canine EPO Preparation and Dosage

Methods used in the production and processing of rcEPO have been previously described.14,15 The resultant rcEPO concentration used to treat dogs in this study was 500 U/mL. The initial dosage of rcEPO was 100 U/kg of body weight SC 3 times/wk. The dose of rcEPO was adjusted at 1- to 4-week intervals to try to achieve and then maintain the Hct in a target range between 35% and 45%. We chose this target range to allow substantial increase in Hct to ameliorate clinical signs of anemia while minimizing possible development of polycythemia. As the study progressed, dogs enrolled in group 2 were started at higher doses of rcEPO (175200 U/kg of body weight SC 3 times/ wk) because of the poor erythroid response of dogs in that group to the initial dose of 100 U/kg 3 times/wk.

Clinical Assessments
Body weight, clinical evaluation by the participating veterinarian, and subjective impressions of the owner regarding the dogs response to treatment were recorded weekly ( 2 days) for the rst 8 weeks and then monthly ( 7 days) for a total study period of 12 months (group 1) or 6 months (group 2). Indirect systolic blood pressure measurements obtained by automated oscillometryc or Doppler owd were recorded monthly ( 7 days).

Laboratory Assessments
CBCs including percentage of reticulocytes, serum iron concentrations, and unsaturated iron-binding capacity were performed before initiating rcEPO treatment and at monthly intervals. Hct (%), erythrocyte number ( 106/ L), and percentage of reticulocytes were determined weekly for the rst 8 weeks. ARC was calculated by multiplying the percentage of reticulocytes by the erythrocyte number ( 106/ L). Total iron binding capacity (TIBC) was determined by summation of serum iron concentration and unsaturated iron-binding capacity. Transferrin saturation was calculated as (serum iron concentration/TIBC) 100. Serum biochemical analyses (sodium, potassium, chloride, blood urea nitrogen [BUN], creatinine, calcium, phosphorus, total protein, albumin, glucose, total bilirubin, cholesterol, and bicarbonate concentrations and alanine transaminase, aspartate transaminase, alkaline phosphatase, and gamma-glutamyl transferase activities) were performed before starting rcEPO treatment and every 2 months thereafter. Week 0 samples were collected within the 2 weeks preceding initiation of rcEPO therapy. For collection of laboratory data after initiating rcEPO treatment, we allowed variability of 2 days for weeks 18 and 7 days for weeks 1252. Multiple laboratories were

Results
Dogs
After they were entered into the study, 7 additional dogs were treated with SC uids, 6 with H2 receptor blockers, 4 with phosphate-binding agents, 4 with metoclopramide HCl, 3 with antihypertensive agents, 1 with sodium bicarbonate, and 1 each with marine oil or vitamin-mineral supplements. In 1 dog, oral androgenic steroids were discon-

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Fig 1. Hematocrit ( ) and absolute reticulocyte count ( ) in group 1 dogs with the anemia of chronic renal failure (A) and group 2 dogs with chronic renal failure and recombinant human erythropoietin-induced red cell aplasia (B) during recombinant canine erythropoietin (rcEPO) treatment. Median rcEPO dose for each group is indicated at the top of its graph. Data points represent median values; number of dogs tested was n 3 for weeks 4052 for group 1 (A) and weeks 424 for group 2 (B). The shaded areas represent the target range for hematocrit (light) and the reference range for absolute reticulocyte number (dark).

tinued upon entry into the study. Ten additional dogs were treated with antibiotics intermittently during the study. All 25 dogs received daily oral supplementation with iron (2 mg elemental iron/kg body weight) starting no later than the onset of rcEPO treatment.

Hematologic Tests
For group 1, median Hct steadily increased during the rst 8 weeks of rcEPO treatment (Fig 1A; Table 1) and was signicantly different (P .0007) from the percentage at week 0 after only 1 week of rcEPO treatment. Median Hct reached our target range of 3545% by week 5 and then was maintained at 35% for the duration of the study. At weeks 8 and 12, the median Hct (for 3 dogs) slightly exceeded our target range. However, with dose adjustments in rcEPO treatment the median Hct remained between 35% and 45% for most of the study (Fig 1A). Median ARC for group 1 was dramatically and signicantly increased (P .0025) from the value at week 0 after 1 week of rcEPO treatment (Fig 1A; Table 1). Median ARC exceeded 80,000 cells/ L by week 2 and peaked at 125,000 cells/ L by

the 3rd week of rcEPO treatment. As median rcEPO dose declined, median ARC also decreased. In contrast, median Hct of group 2 never entered the target range of 3545% despite administration of blood transfusions to 5 of the 6 dogs. Furthermore, median ARC was never 80,000 cells/ L even though median rcEPO doses frequently were greater than those used in group 1 (Fig 1B; Table 1). Median Hct and ARC during rcEPO treatment for group 2 were never signicantly different from those at week 0. For week 0, the median Hct was not signicantly different between the 2 groups, but the median ARC was signicantly and understandably lower (P .01) for the dogs suffering from rhEPO-induced red cell aplasia. Hematocrit and ARC data also are provided from selected individual dogs to illustrate representative responses to rcEPO treatment in groups 1 and 2 (Fig 2). Dogs in group 1 (Fig 2A,B) characteristically demonstrated a rapid and substantial erythrocyte response to rcEPO treatment. The Hct for the dog of Figure 2A was relatively easily maintained within the target range with infrequent adjustments in rcEPO dose, whereas the Hct of the dog of Figure 2B uctuated more widely around the target range. The magnitude of the reticulocyte response also differed between these 2 dogs. In contrast, 4 of 6 dogs in group 2 failed to substantially increase Hct or reticulocyte number with rcEPO treatment, as illustrated by the dog of Figure 2C. The dog in Figure 2D is one of the dogs in group 2 that did develop reticulocytosis and subsequent increased Hct with rcEPO treatment but at doses double those used for group 1. Dogs in this study were classied as having an erythroid response to rcEPO treatment when their ARC increased to 80,000 cells/ L or their Hct increased 50% above their pretreatment Hct without recent administration of blood transfusions or physical or clinicopathologic evidence of profound dehydration. Based on this denition, 95% (18) of the 19 dogs in group 1 and 33% (2) of the 6 dogs in group 2 responded to rcEPO treatment. For group 1, 74% of the dogs responded to rcEPO by week 3, and almost 90% responded by week 5, based on Kaplan-Meier estimates. None of the dogs in group 1 developed an anemia refractory to rcEPO treatment. In the 4 dogs whose Hct initially normalized with rcEPO treatment and then declined with reductions in rcEPO dose, all showed an increase in reticulocyte number, Hct, or both with resumption of the higher rcEPO dose (eg, Fig 2A,B). Median leukocyte counts were between 5.2 and 10.1 103 cells/ L (Table 1) throughout the study for both groups and were not signicantly different between the 2 groups for week 0. The ranges of leukocyte counts for most time points included values of 6.0 103 cells/ L, considered below the lower limit of published reference range for leukocyte numbers in dogs.18 However, differential leukocyte counts only indicated neutropenia (dened as 3,000 neutrophils/ L19) intermittently in 7 dogs (5, group 1; 2, group 2), including 3 dogs with neutropenia before rcEPO administration. In 6 of these 7 dogs, the neutrophil counts normalized during rcEPO treatment. Median platelet counts were between 197 and 441 103 cells/ L throughout the study for both groups (Table 1), but the median count for week 0 was signicantly lower (P .01) for group 2. Com-

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Table 1. Results of hematologic tests in dogs with chronic renal failure (Group 1) and dogs with chronic renal failure and recombinant human erythropoietin-induced red cell aplasia (Group 2) during treatment with recombinant canine erythropoietin (rcEPO).
Reticulocyte ( 103/uL) n 19 18 18 18 18 11 11 11 7 7 7 7 6 5 5 4 3 3 2 2 6 6 6 5 3 3 2 2 2 Median (range) 6.3 74.0 98.8 128.0 58.7 90.0 110.2 39.9 32.8 9.6 24.0 17.3 23.4 11.2 19.6 16.7 31.5 7.9 61.5 28.3 0.0 0.0 0.0 1.0 5.4 28.6 49.2 47.2 49.5 0.0 0.0 0.0 0.0 (049.4) (37.2646.9) (8.0232.2) (10.3209.0) (13.2192.7) (6.2585.9) (49.1302.9) (6.9162.5) (9.165.0) (021.9) (5.679.4) (054.5) (5.563.0) (059.0) (9.264.8) (045.1) (12.472.3) (020.4) (24.498.6) (7.149.5) (07.0) (02.8) (0122.6) (0172.2) (087.8) (0175.7) (098.4) (094.3) (099.0) n 19 12 12 13 18 9 9 9 7 7 7 7 6 5 5 4 3 3 2 2 6 5 5 5 3 3 2 2 2 RBC ( 106/uL) Median (range) 2.7 (1.64.5) 2.8 (2.04.3) 3.7 (2.76.8) 4.2 (3.27.0) 4.8 (2.56.6) 5.6 (4.36.2) 6.2 (4.48.2) 6.7 (3.58.6) 7.1 (6.59.1) 7.3 (6.49.6) 7.4 (5.69.9) 6.3 (4.38.7) 5.9 (3.87.6) 5.6 (4.16.9) 5.4 (3.16.9) 6.6 (4.57.1) 6.3 (6.26.6) 6.5 (5.17.9) 7.0 (5.88.1) 6.3 (5.57.1) 2.2 (1.03.4) 2.3 (1.22.8) 2.1 (1.33.0) 2.0 (1.04.2) 2.7 (2.74.6) 2.5 (2.24.9) 2.3 (2.32.4) 2.1 (2.02.3) 2.7 (2.53.0) 2.5 2.8 2.5 2.1 n 19 12 12 13 18 9 9 9 7 7 7 7 6 5 5 4 3 3 2 2 5 5 5 5 3 3 2 2 2 MCV (fL) Median (range) n MCHC (g/dL) Median (range) n WBC ( 103/uL) Median (range) 7.7 (3.313.7) n 19 Platelet ( 103/uL) Median (range) 441 (133899) n 15

No. of Week Dogs Group 1 0 19 1 19 2 19 3 18 4 18 5 11 6 11 7 11 8 7 12 7 16 7 20 7 24 6 28 5 32 5 36 4 40 3 44 3 48 2 52 2 Group 2 0 1 2 3 4 5 6 7 8 12 16 20 24 Reference rangea 6 6 6 5 3 3 2 2 2 1 1 1 1

Dose rcEPO (U/kg/wk)

Hct (%) Median (range) 16.2 (11.629.0) 21.7 (14.030.0) 23.9 (16.042.6) 28.5 (14.044.4) 32.5 (17.045.3) 37.0 (18.042.0) 38.6 (20.055.2) 40.0 (16.559.8) 46.0 (41.061.7) 46.0 (41.054.2) 44.0 (36.755.1) 38.4 (29.250.4) 36.5 (28.146.0) 38.0 (30.547.0) 36.0 (22.046.0) 43.6 (33.254.0) 46.0 (40.048.1) 40.0 (37.056.1) 47.2 (44.050.4) 45.6 (42.049.1) 15.1 14.7 14.0 14.0 18.0 19.3 17.3 15.5 19.8 16.0 17.5 15.6 15.0 (7.022.8) (7.021.0) (8.030.8) (6.025.0) (17.624.5) (15.025.9) (17.017.6) (13.917.0) (16.523.0)

70 (5177) 19

34.4 (26.038.0) 19

300 300 300 300 300 200 200 200 100 100 100 100 100 100 200 150 150 150 125

(100300) (291300) (200309) (200318) (200300) (200300) (100300) (50300) (0200) (0200) (50200) (70300) (70200) (70200) (70200) (100200) (100150) (100200) (100150)

68 (4977) 18

32.6 (29.035.0) 18

8.9 (4.720.3)

18

413 (1481042) 16

65 63 64 65 67 67 66 70 72 71 69 73

(5872) (5670) (5569) (5678) (5780) (6580) (6473) (6676) (6573) (6273) (6276) (6976)

7 7 7 7 6 5 5 4 3 3 2 2 6

34.0 34.3 33.9 34.5 32.6 31.2 33.2 32.7 32.0 32.0 32.7 31.8

(24.334.5) (28.835.0) (31.035.0) (22.835.2) (31.335.1) (24.235.5) (32.835.2) (30.035.3) (32.033.1) (31.733.3) (30.035.3) (31.032.6)

7 7 7 7 6 5 5 4 3 3 2 2 6

7.7 10.1 9.4 5.2 7.4 8.3 6.6 7.7 7.9 6.3 5.7 6.5

(4.213.4) (3.714.2) (2.319.0) (3.715.4) (4.217.7) (5.310.5) (4.215.3) (5.811.6) (7.912.5) (5.910.4) (3.97.4) (4.88.2)

7 7 7 7 6 4 5 4 3 3 2 2 6

335 268 262 228 315 435 357 338 274 242 197 262

(167754) (101569) (69730) (114562) (114674) (78770) (141746) (172617) (156632) (144500) (103290) (133391)

7 7 6 7 6 5 4 3 3 3 2 2 4

Randolph et al

63 (5167)

35.0 (32.036.0)

6.3 (4.79.2)

292 (220334)

300 413 525 300 300 450 450 450 600 600 600 600

(300600) (300600) (300600) (300600) (300600) (300600) (300600) (300600)

65 (5368)

35.0 (32.636.0)

6.7 (5.87.4)

350 (203375)

71 (6675) 64 62 62 71

33.8 (32.035.5) 36.1 36.7 36.1 31.3

9.5 (8.310.7) 8.6 6.4 5.7 7.9

318 (312323) 354 415 326 413

42.057.0

10.175.9

6.18.5

6374

3237

6.214.4

179483

Hct, hematocrit; RBC, erythrocytes; MCV, mean corpuscular volume; MCHC, mean corpuscular hemoglobin concentration; WBC, leukocytes; n, number of dogs tested. a Established for the Cornell University College of Veterinary Medicine (Ithaca, NY).

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Fig 2. Hematocrit ( ) and absolute reticulocyte count ( ) in individual dogs with chronic renal failure (A and B) or chronic renal failure and recombinant human erythropoietin-induced red cell aplasia (C and D) during recombinant canine erythropoietin (rcEPO) treatment. Dose of rcEPO for each dog is indicated at the top of each graph. The shaded areas represent the target range for hematocrit (light) and the reference range for absolute reticulocyte number (dark). Open arrows indicate administration of blood.

pared with week 0, median platelet counts for dogs in group 1 were not signicantly different at weeks 4 and 8. Only 1 dog (group 1) had platelet counts consistent with thrombocytopenia ( 100 103 cells/ L19 without cytologic evidence of clumping). That dogs platelet numbers uctuated between 69,000 and 172,000 platelets/ L for weeks 844 of rcEPO treatment. Median MCV for dogs in group 1 was signicantly lower (P .03) than that of week 0 for weeks 4, 8, and 12 (Table 1). For week 0, median MCV for group 2 dogs was signicantly lower (P .001) than the corresponding MCV for group 1 dogs. Median mean corpuscular hemoglobin concentration (MCHC) for group 1 dogs was signicantly lower (P .0006) at week 4 than at week 0.

Serum Biochemical Tests

Serum biochemical test results were analyzed for all dogs because pretreatment values were not signicantly different between the 2 groups except for alkaline phosphatase (ALP) activity (P .05). The group 2 median ALP activity for week 0 was 138 U/L, approximately double that of group 1. During rcEPO treatment, differing trends in serum biochemical test results between the 2 groups could not be evaluated because only 2 dogs in group 2 survived to week 8. As expected, median serum BUN, creatinine, and phosphorus concentrations for all dogs were increased at week 0 (Table 2). Median serum BUN, creatinine, and phosphorus concentrations increased mildly but insignicantly from week 0 to week 16. By week 24, the median serum BUN and creatinine concentrations were signicantly increased (P .05) from those at week 0 for surviving dogs, con-

sistent with progression of CRF. Despite some mild deviations from reference ranges, median serum potassium and albumin concentrations for any of the sampled weeks were not signicantly different from their respective pretreatment values. Results of monthly serum iron proles for the 2 groups of dogs are depicted in Table 3; lipemic or hemolyzed serum samples were excluded from analyses. For group 1, median serum iron concentration was below the reference range before administration of rcEPO and decreased further (although not signicantly) during the rst 8 weeks of rcEPO treatment, coinciding with declines in median MCV. For group 2, there were too few dogs tested to detect trends in iron status during rcEPO administration. The week 0 serum iron concentrations were different, but not signicantly so, between the 2 groups, with no overlap in their ranges of values. The higher serum iron concentrations in group 2 dogs may be associated with decreased iron utilization from absent erythropoiesis and blood transfusions received by 4 of the 5 sampled dogs before enrollment in the study.

Bone Marrow Evaluation

Before starting rcEPO treatment, marrow cellularity of the 8 sampled dogs in group 1 was normal (5 of 7 dogs) to decreased (2); cellularity could not be estimated in 1 dog because of lack of bone marrow particles. Megakaryocyte numbers were adequate (410 megakaryocytes/spicule) in 6 dogs and increased (17 megakaryocytes/spicule) in 1 dog. The M : E of between 1.9 : 1 and 19 : 1 in these anemic dogs was consistent with erythroid hypoplasia of chronic renal

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Table 2. Results of serum biochemical tests in dogs with chronic renal failure (Group 1) and dogs with chronic renal failure and recombinant human erythropoietin-induced red cell aplasia (Group 2) during treatment with recombinant canine erythropoietin.
Week 0 Variable Sodium (mEq/L) Potassium (mEq/L) Chloride (mEq/L) BUN (mg/dL) Creatinine (mg/dL) Calcium (mg/dL) Phosphorus (mg/dL) Total protein (g/dL) Albumin (g/dL) Glocose (mg/dL) ALT (U/L) AST (U/L) ALP (U/L) GGT (U/L) Total bilirubin (mg/dL) Cholesterol (mg/dL) Bicarbonate (mEq/L) Median (range) 148 (138154) 5.0 (3.76.8) 113 (99121) 93 (44162) 4.5 (2.711.8) 11.6 (814.2) 7.2 (4.515.9) 5.7 (4.76.9) 2.7 (1.94.2) 104 (87128) 36 (14286) 26 (1354) 77 (14468) 5 (215) 0.2 (0.00.9) 284 (137525) 17 (1322) n 25 25 25 25 25 25 24 25 25 25 25 20 25 10 24 24 7 Week 8 Median (range) 149 (138154) 5.6 (4.06.3) 113 (99116) 89 (32171) 5.0 (3.27.9) 11.2 (8.613.4) 8.7 (3.114.6) 6.3 (5.48.0) 2.9 (2.34.5) 94 (81115) 31 (18205) 32 (1950) 81 (26238) 5 (213) 0.2 (0.10.4) 278 (178466) 16 (1019) n 9 9 9 9 9 9 9 9 9 9 9 7 9 3 9 9 4 Week 16 Median (range) 147 (142150) 5.4 (3.96.2) 113 (100117) 103 (57177) 4.6 (4.010.1) 11.2 (8.212.0) 8.6 (4.112.9) 6.1 (5.56.8) 2.8 (2.53.0) 94 (73118) 24 (20255) 27 (1860) 83 (25161) 5 (015) 0.3 (0.10.5) 326 (211433) 16 (1620) n 7 7 7 8 8 8 8 8 8 8 8 5 8 3 8 8 4 Week 24 Median (range) 145 (135149) 5.5 (4.26.5) 113 (88115) 104 (45232) 5.5 (4.315.3) 10.6 (9.512.5) 7.2 (5.319.2) 5.7 (5.17.0) 2.7 (2.23.0) 96 (90101) 26 (14187) 23 (2147) 58 (18187) 5 (414) 0.1 (0.00.4) 280 (196412) 18 (1422) n 7 7 7 7 7 7 7 7 7 7 7 5 7 3 7 7 4 Week 32 Median (range) 145 (141149) 5.5 (5.46.2) 110 (105115) 82 (73111) 5.0 (4.85.1) 11.0 (10.611.9) 7.1 (5.28.0) 6.3 (6.16.5) 2.8 (2.73.4) 90 (79101) 31 11264) 27 (1048) 54 (23158) 5 (113) 0.1 (0.00.6) 316 (183394) 14 (1315) n 4 4 4 4 4 4 4 4 4 4 4 3 4 3 4 4 2 Week 40 Median (range) 147 (144151) 4.9 (4.56.6) 114 (107114) 108 (89160) 5.3 (5.35.4) 10.9 (10.711.3) 5.9 (5.78.1) 5.8 (5.76.2) 3.0 (2.73.3) 77 (77123) 58 (24170) 32 (2249) 41 (1481) 3 (15) 0.1 (0.10.1) 199 (185386) 15 n 3 3 3 3 3 3 3 3 3 3 3 3 3 2 2 3 1 Week 48 Median (range) 149 (147150) 5.1 (4.65.6) 114 (112115) 125 (70180) 5.2 (4.36.1) 11.3 (11.111.4) 7.7 (5.59.8) 6.1 (5.86.3) 3.2 (3.13.3) 92 (76107) 135 (44225) 31 (2437) 79 (53105) 3 (15) 0.15 (0.10.2) 312 (263361) 13 n 2 3.95.3 2 107117 2 830 2 0.51.3 2 7.212.8 2 3.36.0 2 Referencea Range 142151

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5.67.1 2 3.14.1 2 60120 2 25106 2 1650 2 12122 2 010 2 0.10.2 2 124335 2 1525 1

BUN, blood urea nitrogen; ALT, alanine transaminase; AST, aspartate transaminase; ALP, alkaline phosphatase; GGT, gamma glutamyl-transferase; n, number of dogs tested. a Established for the Cornell University College of Veterinary Medicine (Ithaca, NY).

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Table 3. Results of serum iron proles in dogs with chronic renal failure (group 1) and dogs with chronic renal failure and recombinant human erythropoietin-induced red cell aplasia (group 2) during treatment with recombinant canine erythropoietin.
Iron (mg/dL) Week Group 1 0 4 8 12 16 20 24 28 32 36 40 44 48 52 Group 2 0 4 8 Reference rangea Median (range) 89 (28168) 74 (27200) 75 (57138) 98 (47163) 95 (55239) 135 (64225) 125 (72133) 115 (69488) 86 (66180) 117 (88255) 170 (96244) 83 (53112) 85 101 (95107) 276 (178457) 352 276 98220 n 19 14 8 5 6 6 5 4 5 4 2 2 1 2 5 1 1 TIBC (mg/dL) Median (range) 327 (232435) 312 (180413) 333 (226365) 315 (283337) 315 (212335) 298 (281336) 294 (166345) 313 (275351) 300 (154339) 291 (273322) 308 (270345) 306 (256356) 357 337 (280394) 310 (267457) 425 321 249496 n 16 10 7 5 6 5 5 2 4 4 2 2 1 2 5 1 1 % SAT (%) Median (range) 29 91240) 21 (1048) 22 (1739) 29 (1749) 31 (1888) 46 (30-76) 40 (2575) 39 (2354) 29 (1956) 37 (3293) 54 (3671) 26 (2131) 24 31 (2734) 89 (66100) 83 86 2862 n 16 10 7 5 6 5 5 2 4 4 2 2 1 2 5 1 1

TIBC, total iron-binding capacity; % SAT, percentage of transferrin saturation. a Established for the Cornell University College of Veterinary Medicine (Ithaca, NY).

disease. Marrow iron stores were adequate (2) to increased (5). The increased marrow iron was attributed to decreased iron utilization from decreased erythropoiesis. Three dogs had mild bone marrow lymphocytosis (1016% of total cells), indicating nonspecic antigenic stimulation. Four group 1 dogs had repeat bone marrow evaluations at week 4 of rcEPO treatment. All were characterized by decreased M : E (between 0.7 : 1 and 1.3 : 1) from week 0 values; 2 dogs had erythroid hyperplasia with M : E 1 : 1. Marrow cellularity was normal (1 dog), decreased (1), or increased (2); megakaryocyte numbers were normal, and iron stores were adequate (1) to increased (3). These ndings were consistent with an erythroid response to rcEPO treatment and, in all cases, were associated with increases in Hct. Only 1 dog in group 1 had a bone marrow evaluation performed after 4 weeks. This dog demonstrated an erythroid hyperplasia (M : E of 0.7 : 1, Hct 41%) at 8 weeks, prompting a reduction in rcEPO dose. Fourteen weeks later, the dogs Hct declined to 39% with reticulocytopenia (ARC 0 cells/ L). Bone marrow evaluation at that time revealed an erythroid hypoplasia (M : E of 3.8 : 1) with marrow lymphocytosis (18%). With resumption of an increased rcEPO dose, ARC increased. Bone marrow aspirations on the 6 dogs in group 2 before initiation of rcEPO treatment (week 0) were unsuccessful in 2 dogs, suggesting myelobrosis (later conrmed in 1 dog on subsequent marrow core biopsies). Marrow cellularity was normal (1 of 4 dogs) to decreased (3 dogs). Megakaryocyte numbers were adequate (411 megakaryocytes/spicule). Erythroid precursors were not identied in any dog (M : E of 100 : 1), consistent with red cell apla-

sia. Marrow iron stores were adequate (1 dog) to increased (3). The increased stores were attributed to decreased iron utilization from absent erythropoiesis and recent blood transfusions. Three dogs had mild bone marrow lymphocytosis (1322% of total cells), and 3 had a mild plasmacytosis (59% of total cells), reecting possible immune response to rhEPO. Four dogs had bone marrow evaluations performed after 4 weeks of rcEPO therapy. Two dogs had no evidence of a bone marrow response to rcEPO (ie, erythroid hyperplasia or decreased M : E). The persistent red cell aplasia and bone marrow lymphocytosis in these dogs were associated with continued anemia and reticulocytopenia (eg, Fig 2C). One dog had an erythroid hyperplasia (M : E of 0.45 : 1) at week 4 that was associated with increases in Hct and ARC. Marrow could not be aspirated from the nal dog; therefore, a core biopsy was obtained. This biopsy conrmed myelobrosis (grade IIIII reticulin brosis) and demonstrated erythroid hypoplasia with decreased marrow iron stores. The appearance of erythroid precursors, albeit hypoplastic, suggested a marrow response to rcEPO treatment. Although marrow still could not be aspirated by week 8, another core biopsy revealed erythroid hyperplasia (M : E of 0.9 : 1) associated with increased Hct and reticulocytosis (Fig 2D). However, reticulin brosis (grade II) persisted in the marrow, suggesting that the brosis was secondary to the CRF rather than to rhEPOinduced red cell aplasia.

Other Clinical Parameters

Because of a difference in median body weight between the 2 groups, the median percentage weight change from

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week 0 was evaluated for each group. The dogs in group 1 that survived for 816 weeks showed a trend of weight gain during those weeks that was signicant (P .02) by week 16, even after the 1 dog that was 1 year of age was removed from the analysis. Dogs in group 1 for other weeks of the study and dogs in group 2 throughout the study showed no signicant weight change. Median systolic blood pressure did not change signicantly in either group during rcEPO administration. However, systolic blood pressure of 180 mm Hg did develop in 5 dogs (3, group 1; 2, group 2). For this study, we dened hypertension as a systolic blood pressure repeatedly 180 mm Hg, taken by indirect means in an awake, untrained dog.20,21 Hypertension developed terminally in 2 dogs (group 1), was not treated in 1 dog (group 2), and was treated successfully with amlodipineg in 1 dog (group 2) and unsuccessfully with enalaprilh in 1 dog (group 1). Subjective evaluations during rcEPO treatment in group 1 indicated that appetite improved in 10 (53%) and energy level increased in 14 (74%) of the 19 dogs. These improvements were recorded by owners and veterinarians during the rst few weeks of rcEPO treatment. For group 2 dogs (n 6), improvements in appetite and activity were noted in 4 of the 5 dogs that received blood transfusions and in 2 dogs that developed reticulocytosis and subsequent increased Hct during rcEPO administration. Seizures developed terminally in 1 dog in association with hypertension and bloody diarrhea. However, it was unknown whether the seizures and hypertension were sequelae of progressive renal failure or rcEPO treatment. Pain at injection sites was recorded for 4 dogs during rcEPO treatment, but it was unclear whether these dogs were reacting specically to the rcEPO because 3 were also receiving SC uids. Despite continued rcEPO administration in all 4 dogs, the reported discomfort associated with SC injections resolved. Another dog developed fever and anorexia after the rst dose of rcEPO, but these signs resolved with discontinuation of the rcEPO and did not recur with reinstitution of rcEPO treatment. However, after 3 weeks of rcEPO treatment, that same dog developed a 25-fold increase in serum alanine transaminase (ALT) activity and 8fold increase in ALP activity. With cessation of rcEPO administration, ALT and ALP activities returned to normal. No further resumption of rcEPO therapy was attempted. Lameness during rcEPO treatment was recorded for 3 dogs. However, 2 of the dogs had other conditions (angular limb deformity and traumatic injury) that could account for the lameness, and lameness resolved in both of these dogs despite continued rcEPO administration. The third dog developed lameness terminally. Additional clinical signs that developed during rcEPO treatment but resolved despite continued administration of rcEPO included vulvar blisters (1 dog), acute moist dermatitis (1), and bloody diarrhea (1). Episodic vomiting and poor appetite developed in most dogs during the study but were attributed to uremia. Group 1 dogs survived signicantly longer (P .05) than did group 2 dogs, and group 2 dogs were 3 times more likely to die than were group 1 dogs. Fifty percent of group 1 dogs survived to day 53, whereas 17% of group 2 dogs lived that long.

Discussion
Nonregenerative anemia is an inevitable consequence of CRF.4,8 Although many factors such as reduced erythrocyte life span and gastrointestinal hemorrhage may contribute to the magnitude of the anemia in uremic dogs, the predominant cause is reduced renal production of EPO.4,8 To replace the decient EPO, rhEPO has been administered to dogs with the anemia of CRF. Unfortunately, after an initial erythropoietic response, as many as 50% of these treated dogs ultimately become refractory to therapy and develop a life-threatening anemia caused by rhEPO-induced red cell aplasia.10,12 Dogs apparently develop antibodies against rhEPO because of an 18.7% difference in primary amino acid sequence between human and canine EPO, rendering rhEPO potentially immunogenic in dogs.14 In addition to blocking the efcacy of rhEPO, seroconversion may result in cross-neutralization of any residual endogenous cEPO in uremic dogs, culminating in profound erythroid hypoplasia.1012 The time of erythroid hypoplasia onset in rhEPOtreated uremic dogs has been reported to be as early as 4 weeks after initiation of treatment, but typically this condition is clinically recognized after 2 or 3 months.10,11 The resultant anemia may be more severe than that observed before rhEPO therapy and is refractory to continued rhEPO treatment.12 Erythroid hypoplasia also has been reported after administration of rhEPO to cats1012 and horses.22,23 This problem of immunogenicity severely limits the therapeutic potential of rhEPO in animals and emphasizes the need for species-specic EPO preparations for EPO replacement therapy. Results of the current study indicate that treatment with rcEPO in anemic dogs with CRF (group 1) stimulates erythrocyte production without the immunogenicity problems encountered with rhEPO therapy. Median Hct for dogs in group 1 steadily increased during the rst 8 weeks of rcEPO treatment, reaching the target range of 3545% by week 5 and remaining 35% for the rest of the year of study (Fig 1A; Table 1). Median ARC for group 1 increased signicantly with initial rcEPO dose (100 U/kg SC 3 times/ wk) and declined with dose reductions (Fig 1A; Table 1). Bone marrow cytologic ndings in 4 group 1 dogs after 4 weeks of rcEPO treatment also indicated a stimulation of erythropoiesis. Eighteen (95%) of the 19 dogs in group 1 had an erythroid response to rcEPO characterized by ARC of 80,000 cells/ L or a 50% increase from pretreatment Hct without confounding factors (eg, blood transfusions, profound dehydration). Based on Kaplan-Meier estimates, almost 90% of group 1 dogs had responded to rcEPO by week 5 of treatment. The single dog in group 1 classied as being nonresponsive to rcEPO did have a Hct higher than the baseline value (although a 50% increase) and erythroid hyperplasia on bone marrow evaluation when rcEPO treatment was stopped at week 4 because of possible drug-induced hepatopathy. Four dogs whose Hcts initially normalized with rcEPO treatment and then declined with reductions in rcEPO dose showed an increase in reticulocyte number or Hct with resumption of the higher rcEPO dose (eg, Fig 2A,B). In contrast, rcEPO therapy was not as effective in restoring erythrocyte production in dogs suffering from

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rhEPO-induced red cell aplasia (group 2). In group 2, neither median Hct nor ARC was signicantly different from week 0 during rcEPO treatment (Fig 1B). However, 2 (33%) of the 6 dogs in group 2 did develop erythroid hyperplasia, reticulocytosis, and increased Hcts with rcEPO treatment. Of these rcEPO-responsive dogs, 1 demonstrated reticulocytosis after 2 weeks of rcEPO treatment (300 U/kg/ wk) whereas the other required an additional 4 weeks of rcEPO at 600 U/kg/wk before developing a reticulocyte count of 80,000 cells/ L (Fig 2D). The higher rcEPO dose used in that dog may have been needed to overcome cross-neutralizing antibodies against rhEPO or the existing myelobrosis. In human medicine, uremic patients with severe myelobrosis caused by secondary hyperparathyroidism require a 3 times higher dose of EPO to achieve an adequate Hct.24 Because of the seemingly improved erythroid response of this dog to an increased rcEPO dose, other group 2 dogs were either started at (2 dogs) or increased to (2 dogs) rcEPO doses of 525600 U/kg/wk. Unfortunately, 3 of these dogs were treated for 3 weeks, so their lack of response might be attributable to insufcient length of treatment. For the remaining dog, despite rcEPO administration for 6 months, including 4 months at doses of 600 U/kg/wk, no substantial increase in Hct or reticulocyte number was noted (Fig 2C). The failure of this dog to respond to rcEPO treatment may have been caused by anamnestic production of antibodies to rhEPO elicited either by endogenous cEPO or rcEPO. Recovery from rhEPO-induced red cell aplasia after discontinuation of the drug does occur in dogs with CRF, but the rapidity and the extent of recovery are variable depending in part on the ability of the diseased kidneys to produce adequate amounts of EPO.1012 At best, recovery from rhEPO-induced red cell aplasia in dogs with CRF results in a return of the Hct to the pretreatment value.1012 It is unlikely that the 2 dogs in group 2 categorized as responding to rcEPO treatment recovered spontaneously because their Hcts and reticulocyte numbers after rcEPO administration exceeded their prerhEPO treatment values. Coinciding with improvement in erythroid status, the majority of dogs in group 1 were reported by their owners and veterinarians as demonstrating increased appetite (10 of 19 dogs) and energy level (14 of 19 dogs) despite progression of their renal failure. Owners comments frequently indicated a return of normal activity for their pets (eg, giving the mailman a run for his money again, can jump on the bed by herself again, resumed playtime activity with other dogs). These observations are subjective and we cannot discount a placebo effect. However, dogs in group 1 that survived for 816 weeks did show a trend of weight gain that was signicant (P .02) by week 16. Unexpectedly, some owners remarked that their dogs became dramatically more active during the 1st week, and sometimes even after the 1st dose, of rcEPO treatment, even though no measurable change in Hct had yet occurred. Similar improvements in appetite that predate any erythropoietic response to rhEPO in uremic animals have been described.8 Recent studies have established that human neurons have EPO receptors and that EPO has neuroprotective actions under conditions of hypoxia.25,26 We cannot discount that exogenous EPO may have an effect on the central nervous system

in anemic dogs. For group 2 dogs (n 6), improvements in appetite and activity were noted in 4 of the 5 dogs that received blood transfusions and in the 2 dogs that developed erythroid hyperplasia during rcEPO treatment. These collective observations support the importance of therapeutic intervention to correct the anemia of CRF in dogs to improve their quality of life. In other studies, rhEPO treatment of uremic dogs10,12 and rcEPO administration to clinically normal dogs15 resulted in no changes in mean leukocyte or platelet numbers. However, in rhEPO-treated clinically normal dogs, leukopenia (attributable to neutropenia) and low-normal platelet counts developed with the onset of rhEPO-induced red cell aplasia.15 Such hematologic changes may have been caused by antibody-neutralized EPO activity.15 Similarly, in the dogs of this study, median platelet number at week 0 was signicantly lower for group 2 than for group 1, possibly because of antibody-neutralized EPO activity associated with rhEPO-induced red cell aplasia. However, the exact role that EPO plays in granulopoiesis and thrombopoiesis remains unclear.27,28 Some investigators have reported that recombinant EPO may have stimulatory effects on myeloid and megakaryocytic progenitor cells27 but that EPO-driven erythropoiesis eventually may result in stem cell competition for development between erythrocytes and other blood cells.28 In contrast to those theories, results of the study reported here failed to show any signicant change in median platelet numbers in group 1 during the rst 8 weeks of rcEPO-stimulated erythropoiesis. We cannot discount, however, that thrombocytosis associated with iron deciency may have masked thrombocytopenia in these dogs. A similar phenomenon has been described in rhEPO-treated rats.28 Mild intermittent neutropenia did develop variably in individual dogs in both groups. However, it is unlikely that the neutropenia is a consequence of stem cell competition because there was no signicant correlation between increased ARC (indicative of increased erythropoiesis) and reduced leukocyte numbers. Furthermore, the biologic importance of the neutropenia is questionable because it resolved spontaneously in most cases. Changes in erythrocyte indices for dogs in group 1 during rcEPO treatment were consistent with the development of iron deciency despite daily oral supplementation with iron. Compared with week 0 values, median MCV and MCHC were signicantly lower (P .03 and .0006, respectively) by week 4 of rcEPO treatment. The decline in MCV is even more striking considering that it developed at a time when the characteristically larger reticulocytes were more abundant. Reductions in median serum iron concentration and percentage transferrin saturation for weeks 4 and 8 further support iron deciency in group 1 dogs (Table 3), although chronic disease may have contributed to the decreased values. These ndings are similar to results from an earlier study of clinically normal dogs treated with either rhEPO or rcEPO that also demonstrated decreasing MCV during periods of exogenous EPO-stimulated erythropoiesis despite daily oral supplementation with iron.15 Previous studies in clinically normal humans given rhEPO also have shown that iron deciency develops despite oral supplementation with iron.2931 Relative or functional iron deciency developed in these patients because the macrophages

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could not release iron rapidly enough to meet the increased demand accompanying rhEPO-driven erythropoiesis. Eventually, iron stores become depleted, causing absolute iron deciency. Therefore, despite continued oral supplementation with iron in group 1 dogs, rapid erythrocyte production associated with rcEPO administration may have caused or exacerbated iron deciency resulting in decreased MCV and MCHC. For the 1 dog in group 1 with sequential bone marrow analyses, the development of microcytosis (MCV of 62 fL at week 4 and 58 fL at week 8) despite replete marrow iron stores suggests that relative iron deciency may have been responsible for this dogs declining MCV. In humans, it is doubtful that supplementation with oral iron can ever meet the iron needs of rhEPO-enhanced erythropoiesis, and parenteral supplementation with iron has been advocated.31,32 In recombinant EPO-treated uremic dogs, however, the most effective dose and route of iron supplementation is still unknown. For group 2 dogs, the median MCV at week 0 was signicantly lower (P .001) than the corresponding MCV for group 1. Results of serum iron proles and replete marrow iron stores for group 2 dogs at week 0 did not suggest iron deciency. Rather, the lownormal MCV at week 0 may have reected preexisting iron deciency from previous rhEPO-stimulated erythropoiesis. Iron deciency has been demonstrated previously in uremic dogs during rhEPO treatment12 and may blunt stimulation of erythropoiesis.7,11,2931 In addition to red cell aplasia and iron deciency, other adverse effects that have been reported during rhEPO administration to uremic dogs include polycythemia, seizures, local and systemic allergic reactions, and hypertension.8,1012 In the current study, polycythemia (Hct 60%) developed in 1 rcEPO-treated dog (Fig 2B). However, the dogs Hct never exceeded 62%, and clinical consequences of polycythemia were not evident. With reduction in rcEPO dose, this dogs Hct normalized. The terminal seizures encountered in a single dog and the episodic vomiting and poor appetite that developed in most dogs during the study could be attributed to worsening renal failure rather than adverse effects of rcEPO treatment. One dog did develop increased liver enzyme activities after 3 weeks of rcEPO treatment. Although rcEPO-induced hepatopathy remains a possibility in this dog, similar hepatopathies have not been reported with rhEPO administration to humans or dogs.2,6,8,11,12,15 This dog was later presumptively diagnosed with inammatory bowel disease (IBD), and an association between IBD and liver disease is well established in humans33,34 and commonly observed in dogs.35,36 The development or worsening of hypertension is a concern when rhEPO is used to correct the anemia of CRF in humans, dogs, and cats.12,37,38 With increased erythrocyte production after rhEPO administration, oxygen delivery to tissues is improved, causing decreased vasodilatation and increased peripheral vascular resistance.37 Recent studies also suggest that EPO may cause a Hct-independent vasoconstriction-dependent hypertension, possibly mediated by endothelin or prostanoids.3840 Although median systolic blood pressure in the rcEPO-treated dogs of this study did not change signicantly in either group, 5 (20%) of the 25 dogs did develop systolic blood pressures 180 mm Hg. Two of these dogs (1, group 1; 1, group 2) were already

receiving antihypertension medication. Whether the hypertension was a consequence of rcEPO treatment or a manifestation of the progressive renal failure remains uncertain. In human medicine, clinical studies have proven that rhEPO treatment does not accelerate progression of renal disease despite its vasoconstrictive action.39 Researchers have suggested that rhEPO slows progression of renal disease by correcting hypoxia and exerting antioxidative and antiapoptotic effects.39 Unfortunately, we were unable to compare mortality of the rcEPO-treated dogs of this report with that of similarly affected dogs because of a lack of published survival data for dogs with spontaneous CRF as severe as that encountered in the dogs of this study. In this study, rcEPO stimulated eythrocyte production in dogs with the anemia of CRF during a 1-year treatment period without the red cell aplasia encountered with rhEPO therapy. Benecial effects of rcEPO treatment included improved appetite, increased activity level, and weight gain. Because of the risk of immunogenicity, rhEPO treatment in dogs with CRF generally is reserved for patients with severe anemia (Hct 25%). Because rcEPO appears to represent an effective and safe therapeutic option compared with rhEPO in dogs, treatment with rcEPO could be attempted earlier in the course of CRF before anemia and its attendant clinical signs are so profound. This earlier intervention might allow a longer period of improved quality of life for these dogs. Unfortunately, rcEPO was not as effective in restoring erythrocyte production in dogs with CRF suffering from rhEPO-induced red cell aplasia.

Footnotes
Epogen, Amgen Inc, Thousand Oaks, CA Procrit, Ortho Biotech Inc (Johnson & Johnson), Raritan, NJ c Dinamap vital signs monitor, Critikon, Tampa, FL d Ultrasonic Doppler Flow Detector, Parks Medical Electronics, Aloha, OR e Hitachi 917, Roche Diagnostics, Indianapolis, IN f Advia 120, Bayer Diagnostics, Norwood, MA g Norvasc, Pzer, New York, NY h Enalapril, Merck, Whitehouse Station, NJ
a b

Acknowledgments
This work was supported by the James A. Baker Institute for Animal Health, Pzer Animal Health, the Morris Animal Foundation, and Alumni Unrestricted Gift Funds from the College of Veterinary Medicine at Cornell University. The authors thank Kathryn Saunders and Penelope Ciccone for technical assistance and the following veterinarians for their participation in this multicenter study: Larry Adams (Indiana), Karin Cannizzo (Ohio), Ed Chapman (New York), Terri Cole (New York), Larry Cowgill (California), Beth Crombie (Pennsylvania), Stephen DiBartola (Ohio), Kristen Edwards (Pennsylvania), Michael Friel (New York), Urs Giger (Pennsylvania), Norm Goldstein (New York), Craig Greene (Georgia), Lynn Guptill (Indiana), Steve Hill (California), Jordan Jaeger (Ohio), Gerald Johnson (Florida), Margretta Kethler (Idaho), Linda Kidd (Wisconsin), Sarah Love (Minnesota), Alanna Lowry (Florida),

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Lawrence Monteiro (New York), Amy Morton (Alabama), Noelle Perry (New York), Stacy Riddle (Michigan), Lisa Sepesy (New York), Jennifer Sergeeff (Minnesota), Jerald Shing (New York), Jean Sonneneld (Georgia), Cindy Williams (Alabama), and Patricia Wilson (California).

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